Comparative Genomics of Pseudomonas stutzeri Complex: Taxonomic Assignments and Genetic Diversity

Pseudomonas stutzeri is a species complex with extremely broad phenotypic and genotypic diversity. However, very little is known about its diversity, taxonomy and phylogeny at the genomic scale. To address these issues, we systematically and comprehensively defined the taxonomy and nomenclature for this species complex and explored its genetic diversity using hundreds of sequenced genomes. By combining average nucleotide identity (ANI) evaluation and phylogenetic inference approaches, we identified 123 P. stutzeri complex genomes covering at least six well-defined species among all sequenced Pseudomonas genomes; of these, 25 genomes represented novel members of this species complex. ANI values of ≥∼95% and digital DNA-DNA hybridization (dDDH) values of ≥∼60% in combination with phylogenomic analysis consistently and robustly supported the division of these strains into 27 genomovars (most likely species to some extent), comprising 16 known and 11 unknown genomovars. We revealed that 12 strains had mistaken taxonomic assignments, while 16 strains without species names can be assigned to the species level within the species complex. We observed an open pan-genome of the P. stutzeri complex comprising 13,261 gene families, among which approximately 45% gene families do not match any sequence present in the COG database, and a large proportion of accessory genes. The genome contents experienced extensive genetic gain and loss events, which may be one of the major mechanisms driving diversification within this species complex. Surprisingly, we found that the ectoine biosynthesis gene cluster (ect) was present in all genomes of P. stutzeri species complex strains but distributed at very low frequency (43 out of 9548) in other Pseudomonas genomes, suggesting a possible origin of the ancestors of P. stutzeri species complex in high-osmolarity environments. Collectively, our study highlights the potential of using whole-genome sequences to re-evaluate the current definition of the P. stutzeri complex, shedding new light on its genomic diversity and evolutionary history.

Transcription Factors Pmr1 and Pmr2 Cooperatively Regulate Melanin Biosynthesis, Conidia Development and Secondary Metabolism in Pestalotiopsis microspora

Melanins are the common fungal pigment, which contribute to stress resistance and pathogenesis. However, few studies have explored the regulation mechanism of its synthesis in filamentous fungi. In this study, we identified two transcription factors, Pmr1 and Pmr2, in the filamentous fungus Pestalotiopsis microspora. Computational and phylogenetic analyses revealed that Pmr1 and Pmr2 were located in the gene cluster for melanin biosynthesis. The targeted deletion mutant strain Δpmr1 displayed defects in biosynthesis of conidia pigment and morphological integrity. The deletion of pmr2 resulted in reduced conidia pigment, but the mycelial morphology had little change. Moreover, Δpmr2 produced decreased conidia. RT-qPCR data revealed that expression levels of genes in the melanin biosynthesis gene cluster were downregulated from the loss of Pmr1 and Pmr2. Interestingly, the yield of secondary metabolites in the mutant strains Δpmr1 and Δpmr2 increased, comparing with the wild type, and additionally, Pmr1 played a larger regulatory role in secondary metabolism. Taken together, our results revealed the crucial roles of the transcription factors Pmr1 and Pmr2 in melanin synthesis, asexual development and secondary metabolism in the filamentous fungus P. microspora.

Integrated Omics Approach to Unveil Antifungal Bacterial Polyynes as acetyl-CoA Acetyltransferase Inhibitors

Bacterial polyynes are highly active natural products with a broad spectrum of antimicrobial activities. However, their detailed mechanism of action remains unclear. By integrating comparative genomics, transcriptomics, functional genetics, and metabolomics analysis, we identified a unique polyyne resistance gene, masL (encoding acetyl-CoA acetyltransferase), in the biosynthesis gene cluster of antifungal polyynes (massilin A 1, massilin B 2, collimonin C 3, and collimonin D 4) of Massilia sp. YMA4. Crystallographic analysis indicated that bacterial polyynes serve as covalent inhibitors of acetyl-CoA acetyltransferase. Moreover, we confirmed that the bacterial polyynes disrupted cell membrane integrity and inhibited cell viability of Candida albicans by targeting ERG10, the homolog of MasL. Thus, this study demonstrated that acetyl-CoA acetyltransferase is a potential target for the development of antifungal agents.

A Putative C2H2 Transcription Factor CgTF6, Controlled by CgTF1, Negatively Regulates Chaetoglobosin A Biosynthesis in Chaetomium globosum

Gα signaling pathway as well as the global regulator LaeA were demonstrated to positively regulate the biosynthesis of chaetoglobosin A (ChA), a promising biotic pesticide produced by Chaetomium globosum. Recently, the regulatory function of Zn2Cys6 binuclear finger transcription factor CgcheR that lies within the ChA biosynthesis gene cluster has been confirmed. However, CgcheR was not merely a pathway specific regulator. In this study, we showed that the homologs gene of CgcheR (designated as Cgtf1) regulate ChA biosynthesis and sporulation in C. globosum NK102. More importantly, RNA-seq profiling demonstrated that 1,388 genes were significant differentially expressed as Cgtf1 deleted. Among them, a putative C2H2 transcription factor, named Cgtf6, showed the highest gene expression variation in zinc-binding proteins encoding genes as Cgtf1 deleted. qRT-PCR analysis confirmed that expression of Cgtf6 was significantly reduced in CgTF1 null mutants. Whereas, deletion of Cgtf6 resulted in the transcriptional activation and consequent increase in the expression of ChA biosynthesis gene cluster and ChA production in C. globosum. These data suggested that CgTF6 probably acted as an end product feedback effector, and interacted with CgTF1 to maintain a tolerable concentration of ChA for cell survival.

Heterologous production of novel and rare C30-carotenoids using Planococcus carotenoid biosynthesis genes

Background Members of the genus Planococcus have been revealed to utilize and degrade solvents such as aromatic hydrocarbons and alkanes, and likely to acquire tolerance to solvents. A yellow marine bacterium Planococcus maritimus strain iso-3 was isolated from an intertidal sediment that looked industrially polluted, from the Clyde estuary in the UK. This bacterium was found to produce a yellow acyclic carotenoid with a basic carbon 30 (C30) structure, which was determined to be methyl 5-glucosyl-5,6-dihydro-4,4′-diapolycopenoate. In the present study, we tried to isolate and identify genes involved in carotenoid biosynthesis from this marine bacterium, and to produce novel or rare C30-carotenoids with anti-oxidative activity in Escherichia coli by combinations of the isolated genes. Results A carotenoid biosynthesis gene cluster was found out through sequence analysis of the P. maritimus genomic DNA. This cluster consisted of seven carotenoid biosynthesis candidate genes (orf1–7). Then, we isolated the individual genes and analyzed the functions of these genes by expressing them in E. coli. The results indicated that orf2 and orf1 encoded 4,4′-diapophytoene synthase (CrtM) and 4,4′-diapophytoene desaturase (CrtNa), respectively. Furthermore, orf4 and orf5 were revealed to code for hydroxydiaponeurosporene desaturase (CrtNb) and glucosyltransferase (GT), respectively. By utilizing these carotenoid biosynthesis genes, we produced five intermediate C30-carotenoids. Their structural determination showed that two of them were novel compounds, 5-hydroxy-5,6-dihydro-4,4′-diaponeurosporene and 5-glucosyl-5,6-dihydro-4,4′-diapolycopene, and that one rare carotenoid 5-hydroxy-5,6-dihydro-4,4′-diapolycopene is included there. Moderate singlet oxygen-quenching activities were observed in the five C30-carotenoids including the two novel and one rare compounds. Conclusions The carotenoid biosynthesis genes from P. maritimus strain iso-3, were isolated and functionally identified. Furthermore, we were able to produce two novel and one rare C30-carotenoids in E. coli, followed by positive evaluations of their singlet oxygen-quenching activities.

Complete genome sequence of Bacillus velezensis YYC, a bacterium isolated from the tomato rhizosphere

The Bacillus velezensis YYC strain was isolated from the tomato rhizosphere. In a previous experiment, it increased tomato growth and induced systemic resistance against Ralstonia solanacearum. However, information on its genomic content is lacking. The complete genome sequence of the bacterium was described in this study. The genome size was 3,973,236 bp and consisted of 4,034 genes in total, with a mean G + C content of 46.52%. 86 tRNAs and 27 ri-bosome RNAs were identified. 14 clusters of secondary metabolites were identified. The KEGG database analysis showed that 69 genes were related to quorum sensing, which were important for cross-kingdom communication. In addition, genes involved in promoting plant growth and triggering plant immunity were identified from the genome. Based on digital DNA–DNA hybridizations (dDDH), B. velezensis YYC was the most closely related with B. velezensis FZB42. However, compared with B. velezensis FZB42, the lantipeptide biosynthesis gene cluster was special and only existed in the genome of B. velezensis YYC. The complete genome data of B. velezensis YYC will provide a basis for explanation of its growth-promoting mechanism and biocontrol mechanism.

Genetic fingerprinting and aflatoxin production of Aspergillus section Flavi associated with groundnut in eastern Ethiopia

Background Aspergillus species cause aflatoxin contamination in groundnut kernels, being a health threat in agricultural products and leading to commodity rejection by domestic and international markets. Presence of Aspergillus flavus and A. parasiticus colonizing groundnut in eastern Ethiopia, as well as presence of aflatoxins have been reported, though in this region, no genetic studies have been done of these species in relation to their aflatoxin production. Results In this study, 145 Aspergillus isolates obtained from groundnut kernels in eastern Ethiopia were genetically fingerprinted using 23 Insertion/Deletion (InDel) markers within the aflatoxin-biosynthesis gene cluster (ABC), identifying 133 ABC genotypes. Eighty-four isolates were analyzed by Ultra-Performance Liquid Chromatography (UPLC) for in vitro aflatoxin production. Analysis of genetic distances based on the approximately 85 kb-ABC by Neighbor Joining (NJ), 3D-Principal Coordinate Analysis (3D-PCoA), and Structure software, clustered the isolates into three main groups as a gradient in their aflatoxin production. Group I, contained 98% A. flavus, including L- and non-producers of sclerotia (NPS), producers of B1 and B2 aflatoxins, and most of them collected from the lowland-dry Babile area. Group II was a genetic admixture population of A. flavus (NPS) and A. flavus S morphotype, both low producers of aflatoxins. Group III was primarily represented by A. parasiticus and A. flavus S morphotype isolates both producers of B1, B2 and G1, G2 aflatoxins, and originated from the regions of Darolabu and Gursum. The highest in vitro producer of aflatoxin B1 was A. flavus NPS N1436 (77.98 μg/mL), and the highest producer of aflatoxin G1 was A. parasiticus N1348 (50.33 μg/mL), these isolates were from Gursum and Darolabu, respectively. Conclusions To the best of our knowledge, this is the first study that combined the use of InDel fingerprinting of the ABC and corresponding aflatoxin production capability to describe the genetic diversity of Aspergillus isolates from groundnut in eastern Ethiopia. Three InDel markers, AFLC04, AFLC08 and AFLC19, accounted for the main assignment of individuals to the three Groups; their loci corresponded to aflC (pksA), hypC, and aflW (moxY) genes, respectively. Despite InDels within the ABC being often associated to loss of aflatoxin production, the vast InDel polymorphism observed in the Aspergillus isolates did not completely impaired their aflatoxin production in vitro.

Enhancement of Emodin Production by Medium Optimization and KH2PO4 Supplementation in Submerged Fermentation of Marine-Derived Aspergillus Favipes HN4-13

Emodin is a widely distributed anthraquinone derivative with a variety of biological activities, one that can be efficiently produced by marine-derived fungus Aspergillus favipes HN4-13. However, its relatively low fermentation yield limits further development and pharmaceutical research work. In this study, Plaekett–Burman design and central composite design were adopted to optimize the fermentation conditions of A. favipes HN4-13. Optimal fermentation conditions in a 250-mL Erlenmeyer flask with 50 mL of medium were 59.3 g/L soluble starch, 10 g/L yeast extract paste, 30 g/L seawater salt, 1.04 g/L KH2PO4, 0.05 g/L MgSO4·7H2O, 0.01 g/L FeSO4·7H2O, seed culture 24 h, pH 5, inoculum size 18%, culture temperature 32 °C, and shaking at 160 rpm/min for 7 days. The production of emodin could achieve 132.40 ± 3.09 mg/L, with no significant difference from the predicted value (132.47 mg/L). Furthermore, KH2PO4 supplementation strategy was employed to regulate the mycelial morphology, upregulate the transcriptional level of biosynthesis gene cluster, and enhance emodin production (185.56 ± 4.39 mg/L).

Comparative insights into genome signatures of ferric iron oxide- and anode-stimulated Desulfuromonas spp. strains

Background Halotolerant Fe (III) oxide reducers affiliated in the family Desulfuromonadaceae are ubiquitous and drive the carbon, nitrogen, sulfur and metal cycles in marine subsurface sediment. Due to their possible application in bioremediation and bioelectrochemical engineering, some of phylogenetically close Desulfuromonas spp. strains have been isolated through enrichment with crystalline Fe (III) oxide and anode. The strains isolated using electron acceptors with distinct redox potentials may have different abilities, for instance, of extracellular electron transport, surface recognition and colonization. The objective of this study was to identify the different genomic signatures between the crystalline Fe (III) oxide-stimulated strain AOP6 and the anode-stimulated strains WTL and DDH964 by comparative genome analysis. Results The AOP6 genome possessed the flagellar biosynthesis gene cluster, as well as diverse and abundant genes involved in chemotaxis sensory systems and c-type cytochromes capable of reduction of electron acceptors with low redox potentials. The WTL and DDH964 genomes lacked the flagellar biosynthesis cluster and exhibited a massive expansion of transposable gene elements that might mediate genome rearrangement, while they were deficient in some of the chemotaxis and cytochrome genes and included the genes for oxygen resistance. Conclusions Our results revealed the genomic signatures distinctive for the ferric iron oxide- and anode-stimulated Desulfuromonas spp. strains. These findings highlighted the different metabolic abilities, such as extracellular electron transfer and environmental stress resistance, of these phylogenetically close bacterial strains, casting light on genome evolution of the subsurface Fe (III) oxide reducers.