Backbone Genes Research Articles

Characterization of the Protective Efficacy Against QX Strain of a Recombinant Infectious Bronchitis Virus With H120 Backbone and QX Spike Gene.

Infectious bronchitis virus (IBV) has been prevalent in chicken farms for many years, and its control relies on extensive vaccine administration. The continuous emergence of new variants and the low cross-protection efficiency prompt the development of new vaccines. In this study, we develop a reverse genetics technique based on the classical vaccine strain H120 genome, via in vitro ligation method. Using the H120 genome as the backbone, we constructed the recombinant virus rH120-QX(S) by replacing the H120 S gene with the QX S gene, a prevalent strain in China. Biological characteristics of the rH120-QX(S) virus, such as 50% egg lethal dose (ELD50), 50% egg infectious dose (EID50), dwarf embryo, growth curve, and genetic stability, are measured, which are comparable to the parental virus H120. There are no clinical symptoms and tissue lesions in the trachea and kidney in the rH120-QX(S)-infected specific-pathogen-free (SPF) chickens, demonstrating that this recombinant virus does not confer pathogenicity. Furthermore, protection studies show that there is 100% homologous protection of rH120-QX(S) to the virulent QX strain, as shown by the absence of clinical signs and no lethality. Taken together, our results demonstrate that swapping the S gene onto the H120 genetic backbone is a precise and effective way to produce genetically defined IBV vaccine candidates.

Molecular basis of AR and STK11 genes associated pathogenesis via AMPK pathway and adipocytokine signalling pathway in the development of metabolic disorders in PCOS women

BackgroundOne of the most common hormonal disorders in women of reproductive age is polycystic ovary syndrome (PCOS). In recent years, it has been found that insulin resistance is a common metabolic abnormality in women with PCOS and leads to an elevated risk of type 2 diabetes mellitus. To explore the differentially expressed genes (DEGs) that regulate these kinds of metabolic risks in PCOS women, we chose the gene expression profile of GSE8157 from the gene expression omnibus (GEO) database.ResultsUsing the GEO2R tool, we identified a total of 339 DEGs between the case and the control sample groups. Gene ontology and Kyoto encyclopedia of gene and genome pathway enrichment analysis were subsequently conducted. High connectivity, betweenness centrality, bottleneck centrality, closeness centrality, and radiality measures were used to rank the ten hub genes. Furthermore, the overlap of these genes resulted in the development of two key genes, AR and STK11. The AMPK and adipocytokine signaling pathways are the two main pathways that these DEGs are involved.ConclusionsThe backbone genes, hub genes and pathways identified would assist us in further exploring the molecular basis of developing risk of type 2 diabetes mellitus in PCOS women and thus provide diagnostic or therapeutic clues.

Transcriptional Responses and GCMS Analysis for the Biosynthesis of Pyrethrins and Volatile Terpenes in Tanacetum coccineum.

Natural pyrethrins have been widely used as natural pesticides due to their low mammalian toxicity and environmental friendliness. Previous studies have mainly focused on Tanacetum cinerariifolium, which contains high levels of pyrethrins and volatile terpenes that play significant roles in plant defense and pollination. However, there is little information on T. coccineum due to its lower pyrethrin content and low commercial value. In this study, we measured the transcriptome and metabolites of the leaves (L), flower buds (S1), and fully blossomed flowers (S4) of T. coccineum. The results show that the expression of pyrethrins and precursor terpene backbone genes was low in the leaves, and then rapidly increased in the S1 stage before decreasing again in the S4 stage. The results also show that pyrethrins primarily accumulated at the S4 stage. However, the content of volatile terpenes was consistently low. This perhaps suggests that, despite T. coccineum and T. cinerariifolium having similar gene expression patterns and accumulation of pyrethrins, T. coccineum attracts pollinators via its large and colorful flowers rather than via inefficient and metabolically expensive volatile terpenes, as in T. cinerariifolium. This is the first instance of de novo transcriptome sequencing reported for T. coccineum. The present results could provide insights into pyrethrin biosynthetic pathways and will be helpful for further understanding how plants balance the cost–benefit relationship between plant defense and pollination.

Arginine Methyltransferase PeRmtC Regulates Development and Pathogenicity of Penicilliumexpansum via Mediating Key Genes in Conidiation and Secondary Metabolism.

Penicillium expansum is one of the most common and destructive post-harvest fungal pathogens that can cause blue mold rot and produce mycotoxins in fruit, leading to significant post-harvest loss and food safety concerns. Arginine methylation by protein arginine methyltransferases (PRMTs) modulates various cellular processes in many eukaryotes. However, the functions of PRMTs are largely unknown in post-harvest fungal pathogens. To explore their roles in P. expansum, we identified four PRMTs (PeRmtA, PeRmtB, PeRmtC, and PeRmt2). The single deletion of PeRmtA, PeRmtB, or PeRmt2 had minor or no impact on the P. expansum phenotype while deletion of PeRmtC resulted in decreased conidiation, delayed conidial germination, impaired pathogenicity and pigment biosynthesis, and altered tolerance to environmental stresses. Further research showed that PeRmtC could regulate two core regulatory genes, PeBrlA and PeAbaA, in conidiation, a series of backbone genes in secondary metabolism, and affect the symmetric ω-NG, N’G-dimethylarginine (sDMA) modification of proteins with molecular weights of primarily 16–17 kDa. Collectively, this work functionally characterized four PRMTs in P. expansum and showed the important roles of PeRmtC in the development, pathogenicity, and secondary metabolism of P. expansum.

Complete genome sequence of a novel fish papillomavirus detected in farmed wels catfish (Silurus glanis)

A novel papillomavirus (PV) was detected in farmed wels catfish (Silurus glanis) in Hungary showing clinical signs resembling those of wels catfish herpesvirus disease. The whole genome of Silurus glanis papillomavirus 1 (SgPV1) was identified using next-generation sequencing. The 5,612-bp complete genome contains four predicted protein coding regions (E1, E2, L1, and L2), which seem to have homologues in every PV genome sequenced to date. Five complete fish PV genome sequences are available in the GenBank database. Their genomes range between 5,748 and 6,086 bp and contain the minimal PV backbone genes E1, E2, L2, and L1, unlike PVs of higher vertebrates, which have larger genomes (6.8-8.6 kbp) and additional (onco)genes. Considering the current species demarcation criteria for the family Papillomaviridae, the establishment of a novel species named "Nunpapillomavirus siluri" is proposed for the SgPV1 in a novel genus, "Nunpapillomavirus", in the subfamily Secondpapillomavirinae.

Oncolytic Vaccinia Virus Gene Modification and Cytokine Expression Effects on Tumor Infection, Immune Response, and Killing.

Oncolytic vaccinia viruses have promising efficacy and safety profiles in cancer therapy. Although antitumor activity can be increased by manipulating viral genes, the relative efficacy of individual modifications has been difficult to assess without side-by-side comparisons. This study sought to compare the initial antitumor activity after intravenous administration of five vaccinia virus variants of the same Western Reserve backbone and thymidine kinase gene deletion in RIP-Tag2 transgenic mice with spontaneous pancreatic neuroendocrine tumors. Tumors had focal regions of infection at 5 days after all viruses. Natural killer (NK) cells were restricted to these sites of infection, but CD8+ T cells and tumor cell apoptosis were widespread and varied among the viruses. Antitumor activity of virus VV-A34, bearing amino acid substitution A34K151E to increase viral spreading, and virus VV-IL2v, expressing a mouse IL2 variant (mIL2v) with attenuated IL2 receptor alpha subunit binding, was similar to control virus VV-GFP. However, antitumor activity was significantly greater after virus VV-A34/IL2v, which expressed mIL2v together with A34K151E mutation and viral B18R gene deletion, and virus VV-GMCSF that expressed mouse GM-CSF. Both viruses greatly increased expression of CD8 antigens Cd8a/Cd8b1 and cytotoxicity genes granzyme A, granzyme B, Fas ligand, and perforin-1 in tumors. VV-A34/IL2v led to higher serum IL2 and greater tumor expression of death receptor ligand TRAIL, but VV-GMCSF led to higher serum GM-CSF, greater expression of leukocyte chemokines and adhesion molecules, and more neutrophil recruitment. Together, the results show that antitumor activity is similarly increased by viral expression of GM-CSF or IL2v combined with additional genetic modifications.

Interspecies Genomic Variation and Transcriptional Activeness of Secondary Metabolism-Related Genes in Aspergillus Section Fumigati.

Filamentous fungi produce various bioactive compounds that are biosynthesized by sets of proteins encoded in biosynthesis gene clusters (BGCs). For an unknown reason, many BGCs are transcriptionally silent in laboratory conditions, which has hampered the discovery of novel fungal compounds. The transcriptional reactiveness of fungal secondary metabolism is not fully understood. To gain the comprehensive view, we conducted comparative genomic and transcriptomic analyses of nine closely-related species of Aspergillus section Fumigati (A. fumigatus, A. fumigatiaffinis, A. novofumigatus, A. thermomutatus, A. viridinutans, A. pseudoviridinutans, A. lentulus, A. udagawae, and Neosartorya fischeri). For expanding our knowledge, we newly sequenced genomes of A. viridinutans and A. pseudoviridinutans, and reassembled and reannotated the previously released genomes of A. lentulus and A. udagawae. Between 34 and 84 secondary metabolite (SM) backbone genes were identified in the genomes of these nine respective species, with 8.7-51.2% being unique to the species. A total of 247 SM backbone gene types were identified in the nine fungi. Ten BGCs are shared by all nine species. Transcriptomic analysis using A. fumigatus, A. lentulus, A. udagawae, A. viridinutans, and N. fischeri was conducted to compare expression levels of all SM backbone genes in four different culture conditions; 32-83% of SM backbone genes in these species were not expressed in the tested conditions, which reconfirmed that large part of fungal SM genes are hard to be expressed. The species-unique SM genes of the five species were expressed with lower frequency (18.8% in total) than the SM genes that are conserved in all five species (56%). These results suggest that the expression tendency of BGCs is correlated with their interspecies distribution pattern. Our findings increase understanding of the evolutionary processes associated with the regulation of fungal secondary metabolism.

SARS-CoV-2 vaccine ChAdOx1 nCoV-19 infection of human cell lines reveals low levels of viral backbone gene transcription alongside very high levels of SARS-CoV-2 S glycoprotein gene transcription

BackgroundChAdOx1 nCoV-19 is a recombinant adenovirus vaccine against SARS-CoV-2 that has passed phase III clinical trials and is now in use across the globe. Although replication-defective in normal cells, 28 kbp of adenovirus genes is delivered to the cell nucleus alongside the SARS-CoV-2 S glycoprotein gene.MethodsWe used direct RNA sequencing to analyse transcript expression from the ChAdOx1 nCoV-19 genome in human MRC-5 and A549 cell lines that are non-permissive for vector replication alongside the replication permissive cell line, HEK293. In addition, we used quantitative proteomics to study over time the proteome and phosphoproteome of A549 and MRC5 cells infected with the ChAdOx1 nCoV-19 vaccine.ResultsThe expected SARS-CoV-2 S coding transcript dominated in all cell lines. We also detected rare S transcripts with aberrant splice patterns or polyadenylation site usage. Adenovirus vector transcripts were almost absent in MRC-5 cells, but in A549 cells, there was a broader repertoire of adenoviral gene expression at very low levels. Proteomically, in addition to S glycoprotein, we detected multiple adenovirus proteins in A549 cells compared to just one in MRC5 cells.ConclusionsOverall, the ChAdOx1 nCoV-19 vaccine’s transcriptomic and proteomic repertoire in cell culture is as expected. The combined transcriptomic and proteomics approaches provide a detailed insight into the behaviour of this important class of vaccine using state-of-the-art techniques and illustrate the potential of this technique to inform future viral vaccine vector design.

The chemical profile of activated secondary metabolites by overexpressing LaeA in Aspergillus niger

Although the mechanisms of regulating secondary metabolism by LaeA remains unclear, the synthesis of many secondary metabolites (SMs) in Aspergilli could be activated by LaeA mutation. In our previous sutdy, RNA-seq data has showed that the transcriptional level of many SM backbone genes could be upregulated by overexpressing LaeA. Herein, we analyzed the chemical profile of activated secondary metabolites in the variant of A. niger FGSC A1279 by overexpressing LaeA (OElaeA). 14 compounds were activated in A. niger FGSC A1279 OElaeA variant in the WATM medium. Chemical workup of organic extracts of the culture broth from the A. niger OElaeA mutant identified three pure compounds, flaviolin, orlandin and kotanin. The structures of these compounds were confirmed by HR-ESIMS, 1D/2D NMR, and computer assisted structure elucidation (CASE). Based on homologous alignment and comparison of literatures, the biosynthetic gene cluster (fla) of flaviolin was identified. The in vivo function of the backbone gene, flaA, encoding a multidomain non-reducing polyketide synthase (SAT-KS-AT-PT-ACP), was verified via gene knockout and chemical analysis. Finally, a biosynthetic model for fungal flaviolin was proposed.

In silico assisted identification of peppery aroma compound ‘rotundone’ backbone genes from black pepper

Rotundone, an oxygenated sesquiterpene compound, responsible for the peppery aroma. The importance of the rotundone in the flavor industry warrants search for the precursor genes in plants. We report in this study, the first on the identification of rotundone backbone genes viz., α-guaiene synthase & α-guaiene oxidase in black pepper. We identified the precursor genes of rotundone using berry transcriptome profiling. The metabolite profiling using head space mass spectrometry showed the presence of the direct precursor compounds for rotundone biosynthesis in black pepper berries. The identification of the genes & compounds of the guaiene skeleton is expected to help in bioprospecting of black pepper varieties & also in recombinant production of the aroma compound. Communicated by Ramaswamy H. Sarma Identification of rotundone backbone genes & precursor compounds from Piper nigrum

Plant Species-Dependent Increased Abundance and Diversity of IncP-1 Plasmids in the Rhizosphere: New Insights Into Their Role and Ecology

IncP-1 plasmids, first isolated from clinical specimens (R751, RP4), are recognized as important vectors spreading antibiotic resistance genes. The abundance of IncP-1 plasmids in the environment, previously reported, suggested a correlation with anthropogenic pollution. Unexpectedly, qPCR-based detection of IncP-1 plasmids revealed also an increased relative abundance of IncP-1 plasmids in total community DNA from the rhizosphere of lettuce and tomato plants grown in non-polluted soil along with plant age. Here we report the successful isolation of IncP-1 plasmids by exploiting their ability to mobilize plasmid pSM1890. IncP-1 plasmids were captured from the rhizosphere but not from bulk soil, and a high diversity was revealed by sequencing 14 different plasmids that were assigned to IncP-1β, δ, and ε subgroups. Although backbone genes were highly conserved and mobile elements or remnants as Tn501, IS1071, Tn402, or class 1 integron were carried by 13 of the sequenced IncP-1 plasmids, no antibiotic resistance genes were found. Instead, seven plasmids had a mer operon with Tn501-like transposon and five plasmids contained putative metabolic gene clusters linked to these mobile elements. In-depth sequence comparisons with previously known plasmids indicate that the IncP-1 plasmids captured from the rhizosphere are archetypes of those found in clinical isolates. Our findings that IncP-1 plasmids do not always carry accessory genes in unpolluted rhizospheres are important to understand the ecology and role of the IncP-1 plasmids in the natural environment.

Whole-genome sequence analysis of Salmonella Infantis isolated from raw chicken meat samples and insights into pESI-like megaplasmid

There has been an increase in the number of reports on Salmonella enterica subsp. enterica serovar Infantis (S. Infantis) isolated from animals and humans. Recent studies using whole genome sequencing (WGS) have provided evidence on the likely contribution of a unique conjugative megaplasmid (pESI; ~280 kb) to the dissemination of this serovar worldwide. In the present study, twenty-two unrelated Salmonella strains [S. Infantis (n = 20) and Salmonella 6,7:r:- (n = 2)] and their plasmids were investigated using next generation sequencing technologies (MiSeq and MinION) to unravel the significant expansion of this bacteria in Turkey. Multi-locus sequence typing, plasmid replicons, resistance gene contents as well as phylogenetic relations between strains were determined. According to the WGS data, all S. Infantis possessed the relevant megaplasmid backbone genes and belonged to sequence type 32 (ST32) with the exception of a single novel ST7091. Tetracycline and trimethoprim/sulfamethoxazole resistance were found to be widespread in S. Infantis strains and the resistant strains exclusively carried the tetA, sul1, sul2 and dfrA14 genes. One S. Infantis isolate was also a carrier of the plasmid-mediated ampC via blaCMY-2, gene. Moreover, full genomes of four S. Infantis isolates were reconstructed based on hybrid assembly. All four strains contained large plasmids (240–290 kb) similar to previously published megaplasmid (pESI) and accompanied by several small plasmids. The megaplasmid backbone contained a toxin-antitoxin system, two virulence cassettes and segments associated with heavy metals resistance, while variable regions possessed several antibiotic resistance genes flanked by mobile elements. This study indicated that pESI-like megaplasmid is widely disseminated within the tested S. Infantis strains of chicken meat, warranting further genomic studies on clinical strains from humans and animals to uncover the overall emergence and spread of this serovar.

The large plasmidome of Lactococcus lactis subsp. lactis bv. diacetylactis S50 confers its biotechnological properties

Plasmids are autonomous episomally replicating genetic elements, which carry backbone genes important for the replication and maintenance within their host, and accessory genes that might confer an advantage to their host under specific selective pressure in its ecological niche. The genome of dairy isolate L. lactis subsp. lactis bv. diacetylactis S50 was sequenced using the PacBio SMRT Cell Seq-RSII platform and revealed to possess one of the largest plasmidomes among L. lactis strains studied so far, harboring six plasmids: pS6 (5553 bp), pS7a (7308 bp), pS7b (7266 bp), pS19 (19,027 bp), pS74 (74,256 bp) and pS127 (127,002 bp) in total representing 8.9% of genome size (240,412 bp). Based on predicted plasmid replication proteins and origins it appears that all six plasmids replicate via the theta-type mechanism. The two the largest plasmids (pS74 and pS127), carry a number of genes known to be important for growth and survival in the dairy environment. These genes encode technological functions such as bacteriocin production, protein degradation, magnesium and cobalt/nickel transporters, selenium binding, exopolysaccharides (EPS) production, bacteriophage and stress resistance. Beside genes for replication, the small plasmids (pS6, pS7a, pS7a, and pS19) also carry genes important for mobilization and host survival such as type I restriction-modification (R-M) system, metal transporters, enzymes and transcriptional regulators. All plasmids in S50 strain are mobilizable, containing an oriT sequences, while pS127 is self-conjugative and allows for mobilization of the other plasmids. Small plasmids are prone to structural and segregational instability, while pS127 appeared to be segregationally stable thanks to the possession of two partition systems. The main characteristic of plasmid pS74 is EPS production, while plasmid pS127 is characterized by proteinase and multiple bacteriocins, tra locus, phage abortive systems and metal transporters. In addition to LcnA and LcnB, plasmid pS127 encodes several bacteriocin-pheromone molecules and a new bacteriocin named LcnS50, with narrow spectrum of action limited to lactococci, that has been successfully cloned and heterologously expressed.

Elaborated regulation of griseofulvin biosynthesis in Penicillium griseofulvum and its role on conidiation and virulence

Penicilium griseofulvum, the causal agent of apple blue mold, is able to produce in vitro and on apple a broad spectrum of secondary metabolites (SM), including patulin, roquefortine C and griseofulvin. Among them, griseofulvin is known for its antifungal and antiproliferative activity, and has received interest in many sectors, from medicine to agriculture. The biosynthesis of SM is finely regulated by filamentous fungi and can involve global regulators and pathway specific regulators, which are usually encoded by genes present in the same gene cluster as the backbone gene and tailoring enzymes. In the griseofulvin gene cluster, two putative transcription factors were previously identified, encoded by genes gsfR1 and gsfR2, and their role has been investigated in the present work. Analysis of P. griseofulvum knockout mutants lacking either gene suggest that gsfR2 forms part of a different pathway and gsfR1 exhibits many spectra of action, acting as regulator of griseofulvin and patulin biosynthesis and influencing conidia production and virulence on apple. The analysis of gsfR1 promoter revealed that the regulation of griseofulvin biosynthesis is also controlled by global regulators in response to many environmental stimuli, such as carbon and nitrogen. The influence of carbon and nitrogen on griseofulvin production was further investigated and verified, revealing a complex network of response and confirming the central role of gsfR1 in many processes in P. griseofulvum.

Uncovering the First Atypical DS-1-like G1P[8] Rotavirus Strains That Circulated during Pre-Rotavirus Vaccine Introduction Era in South Africa.

Emergence of DS-1-like G1P[8] group A rotavirus (RVA) strains during post-rotavirus vaccination period has recently been reported in several countries. This study demonstrates, for the first time, rare atypical DS-1-like G1P[8] RVA strains that circulated in 2008 during pre-vaccine era in South Africa. Rotavirus positive samples were subjected to whole-genome sequencing. Two G1P[8] strains (RVA/Human-wt/ZAF/UFS-NGS-MRC-DPRU1971/2008/G1P[8] and RVA/Human-wt/ZAF/UFS-NGS-MRC-DPRU1973/2008/G1P[8]) possessed a DS-1-like genome constellation background (I2-R2-C2-M2-A2-N2-T2-E2-H2). The outer VP4 and VP7 capsid genes of the two South African G1P[8] strains had the highest nucleotide (amino acid) nt (aa) identities of 99.6–99.9% (99.1–100%) with the VP4 and the VP7 genes of a locally circulating South African strain, RVA/Human-wt/ZAF/MRC-DPRU1039/2008/G1P[8]. All the internal backbone genes (VP1–VP3, VP6, and NSP1-NSP5) had the highest nt (aa) identities with cognate internal genes of another locally circulating South African strain, RVA/Human-wt/ZAF/MRC-DPRU2344/2008/G2P[6]. The two study strains emerged through reassortment mechanism involving locally circulating South African strains, as they were distinctly unrelated to other reported atypical G1P[8] strains. The identification of these G1P[8] double-gene reassortants during the pre-vaccination period strongly supports natural RVA evolutionary mechanisms of the RVA genome. There is a need to maintain long-term whole-genome surveillance to monitor such atypical strains.

Detection of three independently-generated DS-1-like G9P[8] reassortant rotavirus A strains during the G9P[8] dominance in Vietnam, 2016–2018

A rapid increase and dominance of G9P[8] Rotavirus A strains occurred in northern Vietnam between 2016 and 2018, during which period there appeared three G9P[8] strains possessing short RNA patterns. To understand how the first-ever G9P[8] strains possessing short RNA patterns were formed, next generation sequencing technology was used to examine the whole genomes of the three strains, i.e., RVA/Human-wt/VNM/RVN16.1024/2016/G9P[8], RVA/Human-wt/VNM/RVN17.0879/2017/G9P[8], and RVA/Human-wt/VNM/RVN18.0197/2018/G9P[8], and those of seven representative G9P[8] strains possessing long RNA patterns. The VP7 genes of the short and long G9P[8] strains were > 99% identical, indicating that the origin was in the co-circulating, dominant, long G9P[8] strains. On the other hand, the VP4 genes likely derived from recently-emerging G1/G3/G8P[8] strains possessing the DS-1 backbone. At the lineage level, however, the backbone genes of any one strain differed from that of the other two in the VP1, VP3 or NSP4 gene. Moreover, even at the nucleotide sequence level of the backbone genes belonging to the same lineage, the identities between the three strains were lower than those expected for the strains deriving from an immediate, common ancestor. Thus, the three strains were likely formed by independent reassortment events in which the VP7 gene of the currently dominant G9P[8] strains was incorporated into co-circulating G1/G3/G8P[8] strains possessing similar yet distinct DS-1-like backbone genes. The observation that all of the three reassortant G9P[8] strains were detected only once among the prevalent, ordinary G9P[8] strains suggests that acquisition of the DS-1-like backbone genes unlikely provided selective advantage over the parental Wa-like G9P[8] strains.

Requirement of LaeA, VeA, and VelB on Asexual Development, Ochratoxin A Biosynthesis, and Fungal Virulence in Aspergillus ochraceus.

Aspergillus ochraceus is reported to be the major contributor of ochratoxin A (OTA), classified as one of the possible human carcinogen (group 2B) by the International Agency for Research on Cancer. The heterotrimeric velvet complex proteins, LaeA/VeA/VelB, have been most studied in fungi to clarify the relation between light-dependent morphology and secondary metabolism. To explore possible genetic targets to control OTA contamination, we have identified laeA, veA, and velB in A. ochraceus. The loss of laeA, veA, and velB yielded mutants with differences in vegetative growth and conidial production. Especially, ΔlaeA almost lost the ability to generate conidiaphore under dark condition. The deletion of laeA, veA, and velB drastically reduced the production of OTA. The wild-type A. ochraceus produced about 1 and 7 μg/cm2 OTA under light and dark conditions on media, whereas the three gene deletion mutants produced less than 20 ng/cm2 OTA, which was correlated with a down regulation of OTA biosynthetic genes. Pathogenicity studies of ΔlaeA, ΔveA, and ΔvelB showed their reduction in disease severity in pears. Furthermore, 66.1% of the backbone genes in secondary metabolite gene cluster were significantly regulated, among which 81.6% were downregulated. Taking together, these results revealed that velvet complex proteins played crucial roles in asexual development, secondary metabolism, and fungal virulence in A. ochraceus.

Genome-wide DNA methylation analysis of Metarhizium anisopliae during tick mimicked infection condition

BackgroundThe Metarhizium genus harbors important entomopathogenic fungi. These species have been widely explored as biological control agents, and strategies to improve the fungal virulence are under investigation. Thus, the interaction between Metarhizium species and susceptible hosts have been explored employing different methods in order to characterize putative virulence determinants. However, the impact of epigenetic modulation on the infection cycle of Metarhizium is still an open topic. Among the different epigenetic modifications, DNA methylation of cytosine bases is an important mechanism to control gene expression in several organisms. To better understand if DNA methylation can govern Metarhizium-host interactions, the genome-wide DNA methylation profile of Metarhizium anisopliae was explored in two conditions: tick mimicked infection and a saprophytic-like control.ResultsUsing a genome wide DNA methylation profile based on bisulfite sequencing (BS-Seq), approximately 0.60% of the total cytosines were methylated in saprophytic-like condition, which was lower than the DNA methylation level (0.89%) in tick mimicked infection condition. A total of 670 mRNA genes were found to be putatively methylated, with 390 mRNA genes uniquely methylated in the tick mimicked infection condition. GO terms linked to response to stimuli, cell wall morphogenesis, cytoskeleton morphogenesis and secondary metabolism biosynthesis were over-represented in the tick mimicked infection condition, suggesting that energy metabolism is directed towards the regulation of genes associated with infection. However, recognized virulence determinants known to be expressed at distinct infection steps, such as the destruxin backbone gene and the collagen-like protein gene Mcl1, were found methylated, suggesting that a dynamic pattern of methylation could be found during the infectious process. These results were further endorsed employing RT-qPCR from cultures treated or not with the DNA methyltransferase inhibitor 5-Azacytidine.ConclusionsThe set of genes here analyzed focused on secondary metabolites associated genes, known to be involved in several processes, including virulence. The BS-Seq pipeline and RT-qPCR analysis employing 5-Azacytidine led to identification of methylated virulence genes in M. anisopliae. The results provided evidences that DNA methylation in M. anisopliae comprises another layer of gene expression regulation, suggesting a main role of DNA methylation regulating putative virulence determinants during M. anisopliae infection cycle.

SGI0, a relative of Salmonella genomic islands SGI1 and SGI2, lacking a class 1 integron, found in Proteus mirabilis

Several groups of integrative mobilizable elements (IMEs) that harbour a class 1 integron carrying antibiotic resistance genes have been found at the 3′-end of the chromosomal trmE gene. Here, a new IME, designated SGI0, was found in trmE in the sequenced and assembled genome of a French clinical, multiply antibiotic resistant Proteus mirabilis strain, Pm1LENAR. SGI0 shares the same gene content as the backbones of SGI1 and SGI2 (overall 97.6% and 97.7% nucleotide identity, respectively) but it lacks a class 1 integron. However, SGI0 is a mosaic made up of segments with >98.5% identity to SGI1 and SGI2 interspersed with segments sharing 74–95% identity indicating that further diverged backbone types exist and that recombination between them is occurring. The structure of SGI1-V, here re-named SGI-V, which lacks two SGI1 (S023 and S024) backbone genes and includes a group of additional genes in the backbone, was re-examined. In regions shared with SGI1, the backbones shared 97.3% overall identity with the differences distributed in patches with various levels of identity. The class 1 integron is also in a slightly different position with the target site duplication AAATT instead of ACTTG for SGI1 and variants, indicating that it was acquired independently. The Pm1LENAR resistance genes are in the chromosome, in Tn7 and an ISEcp1-mobilised segment.

Do Organic Substrates Drive Microbial Community Interactions in Arctic Snow?

The effect of nutrients on microbial interactions, including competition and collaboration, has mainly been studied in laboratories, but their potential application to complex ecosystems is unknown. Here, we examined the effect of changes in organic acids among other parameters on snow microbial communities in situ over 2 months. We compared snow bacterial communities from a low organic acid content period to that from a higher organic acid period. We hypothesized that an increase in organic acids would shift the dominant microbial interaction from collaboration to competition. To evaluate microbial interactions, we built taxonomic co-variance networks from OTUs obtained from 16S rRNA gene sequencing. In addition, we tracked marker genes of microbial cooperation (plasmid backbone genes) and competition (antibiotic resistance genes) across both sampling periods in metagenomes and metatranscriptomes. Our results showed a decrease in the average connectivity of the network during late spring compared to the early spring that we interpreted as a decrease of cooperation. This observation was strengthened by the significantly more abundant plasmid backbone genes in the metagenomes from the early spring. The modularity of the network from the late spring was also found to be higher than the one from the early spring, which is another possible indicator of increased competition. Antibiotic resistance genes were significantly more abundant in the late spring metagenomes. In addition, antibiotic resistance genes were also positively correlated to the organic acid concentration of the snow across both seasons. Snow organic acid content might be responsible for this change in bacterial interactions in the Arctic snow community.