Pathogenicity is associated with population structure in a fungal pathogen of humans.

Aspergillus flavus is a clinically and agriculturally important saprotrophic fungus responsible for severe human infections and extensive crop losses. We analyzed genomic data from 250 (95 clinical and 155 environmental) A. flavus isolates from 9 countries, including 70 newly sequenced clinical isolates, to examine population and pan-genome structure and their relationship to pathogenicity. We identified five A. flavus populations, including a new population, D, corresponding to distinct clades in the genome-wide phylogeny. Strikingly, > 75% of clinical isolates were from population D. Accessory genes, including genes within biosynthetic gene clusters, were significantly more common in some populations but rare in others. Population D was enriched for genes associated with zinc ion binding, lipid metabolism, and certain types of hydrolase activity. In contrast to the major human pathogen Aspergillus fumigatus , A. flavus pathogenicity in humans is strongly associated with population structure, making it a great system for investigating how population-specific genes contribute to pathogenicity.

Draft genomes of Aspergillus nomiae and Aspergillus tamarii isolates from human eye infections.

Fungal species within the genus Aspergillus are an important cause of human diseases, including aspergillosis and keratitis. We present draft genomes of Aspergillus tamarii and A. nomiae isolated from keratitis patients in a U.S. tertiary care hospital.

A comprehensive map of hotspots of de novo telomere addition in Saccharomyces cerevisiae.

Telomere healing occurs when telomerase, normally restricted to chromosome ends, acts upon a double-strand break to create a new, functional telomere. De novo telomere addition (dnTA) on the centromere-proximal side of a break truncates the chromosome but, by blocking resection, may allow the cell to survive an otherwise lethal event. We previously identified several sequences in the baker's yeast, Saccharomyces cerevisiae, that act as hotspots of dnTA [termed Sites of Repair-associated Telomere Addition (SiRTAs)], but the distribution and functional relevance of SiRTAs is unclear. Here, we describe a high-throughput sequencing method to measure the frequency and location of telomere addition within sequences of interest. Combining this methodology with a computational algorithm that identifies SiRTA sequence motifs, we generate the first comprehensive map of telomere-addition hotspots in yeast. Putative SiRTAs are strongly enriched in subtelomeric regions where they may facilitate formation of a new telomere following catastrophic telomere loss. In contrast, outside of subtelomeres, the distribution and orientation of SiRTAs appears random. Since truncating the chromosome at most SiRTAs would be lethal, this observation argues against selection for these sequences as sites of telomere addition per se. We find, however, that sequences predicted to function as SiRTAs are significantly more prevalent across the genome than expected by chance. Sequences identified by the algorithm bind the telomeric protein Cdc13, raising the possibility that association of Cdc13 with single-stranded regions generated during the response to DNA damage may facilitate DNA repair more generally.

A comprehensive map of hotspots of de novo telomere addition in Saccharomyces cerevisiae.

Telomere healing occurs when telomerase, normally restricted to chromosome ends, acts upon a double-strand break to create a new, functional telomere. De novo telomere addition on the centromere-proximal side of a break truncates the chromosome but, by blocking resection, may allow the cell to survive an otherwise lethal event. We previously identified several sequences in the baker’s yeast, Saccharomyces cerevisiae , that act as hotspots of de novo telomere addition (termed Sites of Repair-associated Telomere Addition or SiRTAs), but the distribution and functional relevance of SiRTAs is unclear. Here, we describe a high-throughput sequencing method to measure the frequency and location of telomere addition within sequences of interest. Combining this methodology with a computational algorithm that identifies SiRTA sequence motifs, we generate the first comprehensive map of telomere-addition hotspots in yeast. Putative SiRTAs are strongly enriched in subtelomeric regions where they may facilitate formation of a new telomere following catastrophic telomere loss. In contrast, outside of subtelomeres, the distribution and orientation of SiRTAs appears random. Since truncating the chromosome at most SiRTAs would be lethal, this observation argues against selection for these sequences as sites of telomere addition per se. We find, however, that sequences predicted to function as SiRTAs are significantly more prevalent across the genome than expected by chance. Sequences identified by the algorithm bind the telomeric protein Cdc13, raising the possibility that association of Cdc13 with single-stranded regions generated during the response to DNA damage may facilitate DNA repair more generally.

Genomic and Phenotypic Trait Variation of the Opportunistic Human Pathogen Aspergillus flavus and Its Close Relatives.

Fungal diseases affect millions of humans annually, yet fungal pathogens remain understudied. The mold Aspergillus flavus can cause both aspergillosis and fungal keratitis infections, but closely related species are not considered clinically relevant. To study the evolution of A. flavus pathogenicity, we examined genomic and phenotypic traits of two strains of A. flavus and three closely related species, Aspergillus arachidicola (two strains), Aspergillus parasiticus (two strains), and Aspergillus nomiae (one strain). We identified >3,000 orthologous proteins unique to A. flavus, including seven biosynthetic gene clusters present in A. flavus strains and absent in the three nonpathogens. We characterized secondary metabolite production for all seven strains under two clinically relevant conditions, temperature and salt concentration. Temperature impacted metabolite production in all species, whereas salinity did not affect production of any species. Strains of the same species produced different metabolites. Growth under stress conditions revealed additional heterogeneity within species. Using the invertebrate fungal disease model Galleria mellonella, we found virulence of strains of the same species varied widely; A. flavus strains were not more virulent than strains of the nonpathogens. In a murine model of fungal keratitis, we observed significantly lower disease severity and corneal thickness for A. arachidicola compared to other species at 48 h postinfection, but not at 72 h. Our work identifies variations in key phenotypic, chemical, and genomic attributes between A. flavus and its nonpathogenic relatives and reveals extensive strain heterogeneity in virulence that does not correspond to the currently established clinical relevance of these species. IMPORTANCE Aspergillus flavus is a filamentous fungus that causes opportunistic human infections, such as aspergillosis and fungal keratitis, but its close relatives are considered nonpathogenic. To begin understanding how this difference in pathogenicity evolved, we characterized variation in infection-relevant genomic, chemical, and phenotypic traits between strains of A. flavus and its relatives. We found extensive variation (or strain heterogeneity) within the pathogenic A. flavus as well as within its close relatives, suggesting that strain-level differences may play a major role in the ability of these fungi to cause disease. Surprisingly, we also found that the virulence of strains from species not considered to be pathogens was similar to that of A. flavus in both invertebrate and murine models of disease. These results contrast with previous studies on Aspergillus fumigatus, another major pathogen in the genus, for which significant differences in infection-relevant chemical and phenotypic traits are observed between closely related pathogenic and nonpathogenic species.

Draft Genome Sequence of an Aspergillus Strain Isolated from a Honey Bee Pupa.

Insect-associated fungi play an important role in wild and agricultural communities. We present a draft genome sequence of an entomopathogenic strain from the fungal genus Aspergillus, isolated from a honey bee pupa.

Complete Mitochondrial Genome Sequences of Nine Aspergillus flavus Strains.

Nuclear genome sequences incompletely characterize the genomic content and thus the genetic diversity of fungal species. Here, we present the complete mitochondrial genome sequences of nine Aspergillus flavus strains, providing useful information for inter- and intraspecific analyses.

Genomes of Gut Bacteria from Nasonia Wasps Shed Light on Phylosymbiosis and Microbe-Assisted Hybrid Breakdown.

Phylosymbiosis is a cross-system trend whereby microbial community relationships recapitulate the host phylogeny. In Nasonia parasitoid wasps, phylosymbiosis occurs throughout development, is distinguishable between sexes, and benefits host development and survival. Moreover, the microbiome shifts in hybrids as a rare Proteus bacterium in the microbiome becomes dominant. The larval hybrids then catastrophically succumb to bacterium-assisted lethality and reproductive isolation between the species. Two important questions for understanding phylosymbiosis and bacterium-assisted lethality in hybrids are (i) do the Nasonia bacterial genomes differ from other animal isolates and (ii) are the hybrid bacterial genomes the same as those in the parental species? Here, we report the cultivation, whole-genome sequencing, and comparative analyses of the most abundant gut bacteria in Nasonia larvae, Providencia rettgeri and Proteus mirabilis Characterization of new isolates shows Proteus mirabilis forms a more robust biofilm than Providencia rettgeri and that, when grown in coculture, Proteus mirabilis significantly outcompetes Providencia rettgeri Providencia rettgeri genomes from Nasonia are similar to each other and more divergent from pathogenic, human associates. Proteus mirabilis from Nasonia vitripennis, Nasonia giraulti, and their hybrid offspring are nearly identical and relatively distinct from human isolates. These results indicate that members of the larval gut microbiome within Nasonia are most similar to each other, and the strain of the dominant Proteus mirabilis in hybrids is resident in parental species. Holobiont interactions between shared, resident members of the wasp microbiome and the host underpin phylosymbiosis and hybrid breakdown.IMPORTANCE Animal and plant hosts often establish intimate relationships with their microbiomes. In varied environments, closely related host species share more similar microbiomes, a pattern termed phylosymbiosis. When phylosymbiosis is functionally significant and beneficial, microbial transplants between host species and host hybridization can have detrimental consequences on host biology. In the Nasonia parasitoid wasp genus, which contains a phylosymbiotic gut community, both effects occur and provide evidence for selective pressures on the holobiont. Here, we show that bacterial genomes in Nasonia differ from other environments and harbor genes with unique functions that may regulate phylosymbiotic relationships. Furthermore, the bacteria in hybrids are identical to those in parental species, thus supporting a hologenomic tenet that the same members of the microbiome and the host genome impact phylosymbiosis, hybrid breakdown, and speciation.

Production of itaconic acid from alkali pretreated lignin by dynamic two stage bioconversion.

Expanding the portfolio of products that can be made from lignin will be critical to enabling a viable bio-based economy. Here, we engineer Pseudomonas putida for high-yield production of the tricarboxylic acid cycle-derived building block chemical, itaconic acid, from model aromatic compounds and aromatics derived from lignin. We develop a nitrogen starvation-detecting biosensor for dynamic two-stage bioproduction in which itaconic acid is produced during a non-growth associated production phase. Through the use of two distinct itaconic acid production pathways, the tuning of TCA cycle gene expression, deletion of competing pathways, and dynamic regulation, we achieve an overall maximum yield of 56% (mol/mol) and titer of 1.3 g/L from p-coumarate, and 1.4 g/L titer from monomeric aromatic compounds produced from alkali-treated lignin. This work illustrates a proof-of-principle that using dynamic metabolic control to reroute carbon after it enters central metabolism enables production of valuable chemicals from lignin at high yields by relieving the burden of constitutively expressing toxic heterologous pathways.

Metabolism of syringyl lignin-derived compounds in Pseudomonas putida enables convergent production of 2-pyrone-4,6-dicarboxylic acid.

Valorization of lignin, an abundant component of plant cell walls, is critical to enabling the lignocellulosic bioeconomy. Biological funneling using microbial biocatalysts has emerged as an attractive approach to convert complex mixtures of lignin depolymerization products to value-added compounds. Ideally, biocatalysts would convert aromatic compounds derived from the three canonical types of lignin: syringyl (S), guaiacyl (G), and p-hydroxyphenyl (H). Pseudomonas putida KT2440 (hereafter KT2440) has been developed as a biocatalyst owing in part to its native catabolic capabilities but is not known to catabolize S-type lignin-derived compounds. Here, we demonstrate that syringate, a common S-type lignin-derived compound, is utilized by KT2440 only in the presence of another energy source or when vanAB was overexpressed, as syringate was found to be O-demethylated to gallate by VanAB, a two-component monooxygenase, and further catabolized via extradiol cleavage. Unexpectedly, the specificity (kcat/KM) of VanAB for syringate was within 25% that for vanillate and O-demethylation of both substrates was well-coupled to O2 consumption. However, the native KT2440 gallate-cleaving dioxygenase, GalA, was potently inactivated by 3-O-methylgallate. To engineer a biocatalyst to simultaneously convert S-, G-, and H-type monomers, we therefore employed VanAB from Pseudomonas sp. HR199, which has lower activity for 3MGA, and LigAB, an extradiol dioxygenase able to cleave protocatechuate and 3-O-methylgallate. This strain converted 93% of a mixture of lignin monomers to 2-pyrone-4,6-dicarboxylate, a promising bio-based chemical. Overall, this study elucidates a native pathway in KT2440 for catabolizing S-type lignin-derived compounds and demonstrates the potential of this robust chassis for lignin valorization.

Complete chloroplast genome comparisons for Pityopsis (Asteraceae).

Pityopsis includes several regionally and one federally endangered species of herbaceous perennials. Four species are highly localized, including the federally endangered P. ruthii. The genus includes several ploidy levels and interesting ecological traits such as drought tolerance and fire-dependent flowering. Results from previous cladistic analyses of morphology and from initial DNA sequence studies did not agree with one another or with the infrageneric taxonomic classification, with the result that infrageneric relationships remain unresolved. We sequenced, assembled, and compared the chloroplast (cp) genomes of 12 species or varieties of Pityopsis to better understand generic evolution. A reference cp genome 152,569 bp in length was assembled de novo from P. falcata. Reads from other sampled species were then aligned to the P. falcata reference and individual chloroplast genomes were assembled for each, with manual gapfilling and polishing. After removing the duplicated second inverted region, a multiple sequence alignment of the cp genomes was used to construct a maximum likelihood (ML) phylogeny for the twelve cp genomes. Additionally, we constructed a ML phylogeny from the nuclear ribosomal repeat region after mapping reads to the Helianthus annuus region. The chloroplast phylogeny supported two clades. Previously proposed clades and taxonomic sections within the genus were largely unsupported by both nuclear and chloroplast phylogenies. Our results provide tools for exploring hybridity and examining the physiological and genetic basis for drought tolerance and fire-dependent flowering. This study will inform breeding and conservation practices, and general knowledge of evolutionary history, hybridization, and speciation within Pityopsis.

Complete Genome Sequences of Four Natural Pseudomonas Isolates That Catabolize a Wide Range of Aromatic Compounds Relevant to Lignin Valorization.

Many soil microorganisms have evolved catabolic strategies to utilize phenolic compounds arising from depolymerized lignin. We report the complete genome sequences of four Pseudomonas sp. isolates that demonstrated robust growth on a wide range of aromatic monomers and dimers that are relevant to the valorization of lignin into value-added chemicals.

Revised Transcriptome-Based Gene Annotation for Aspergillus flavus Strain NRRL 3357.

Aspergillus flavus is an agriculturally and medically important filamentous fungus that produces mycotoxins, including aflatoxins, which are potent carcinogens. Here, we generated short- and long-read transcript sequence data from the growth of A. flavus strain NRRL 3357 under both typical and stress conditions to produce a new annotation of its genome.

Methylome and Complete Genome Sequence of Parageobacillus toebii DSM 14590T, a Thermophilic Bacterium.

Here, we present the first complete genome assembly of the thermophilic bacterium Parageobacillus toebii DSM 14590T The P. toebii DSM 14590T genome consists of a 3,270,071-bp circular chromosome and a 52,989-bp native plasmid.

Complete Genome Sequence of Caloramator sp. Strain E03, a Novel Ethanologenic, Thermophilic, Obligately Anaerobic Bacterium.

Here, we report the complete genome sequence of Caloramator sp. strain E03, an anaerobic thermophile that was isolated from a hot spring within the Rabbit Creek area of Yellowstone National Park. The assembly contains a single 2,984,770-bp contig with a G+C content of 31.3% and is predicted to encode 2,678 proteins.

Complete Genome Sequences of Two Megasphaera elsdenii Strains, NCIMB 702410 and ATCC 25940.

Here, we report the complete genome sequences of two Megasphaera elsdenii strains, ATCC 25940 and NCIMB 702410. M. elsdenii is an anaerobic bacterium capable of producing butanoate and hexanoate and is a member of the Negativicutes.

Complete Genome Sequence of Salinisphaera sp. Strain LB1, a Moderately Halo-Acidophilic Bacterium Isolated from Lake Brown, Western Australia.

Salinisphaera sp. strain LB1 was isolated from Lake Brown, Western Australia, surface water enriched at pH 4.0 and with 5% (wt/vol) NaCl. The complete genome sequence is presented in this report.

Population Structure and Genetic Diversity Within the Endangered Species Pityopsis ruthii (Asteraceae).

Pityopsis ruthii (Ruth's golden aster) is a federally endangered herbaceous perennial endemic to the Hiwassee and Ocoee Rivers in southeastern Tennessee, United States. Comprehensive genetic studies providing novel information to conservationists for preservation of the species are lacking. Genetic variation and gene flow were evaluated for 814 individuals from 33 discrete locations using polymorphic microsatellites: seven chloroplast and twelve nuclear. A total of 198 alleles were detected with the nuclear loci and 79 alleles with the chloroplast loci. Gene flow was estimated, with the Hiwassee River (Nm = 2.16; FST = 0.15) showing higher levels of gene flow and lower levels of population differentiation than the Ocoee River (Nm = 1.28; FST = 0.19). Population structure was examined using Bayesian cluster analyses. Nuclear and chloroplast analyses were incongruent. From the chloroplast microsatellites, three clusters were identified; all were present in sampling sites at both rivers, indicating a lack of allele fixation along rivers. Nuclear markers revealed two clusters and separated by river. When the Hiwassee River locations were analyzed, four clusters were identified for both the chloroplast and nuclear microsatellites, though the individuals clustered differently. Analysis of the Ocoee River revealed two clusters for the chloroplast microsatellites and three for the nuclear microsatellites. We recommend P. ruthii be managed as four populations for the Hiwassee River and three populations for the Ocoee River. Our results provide critical genetic information for P. ruthii that can be used for species management decisions to drive future population augmentation/reintroduction and ex situ conservation efforts.

Complete Genome Sequence of Industrial Dairy Strain Streptococcus thermophilus DGCC 7710.

We report here the complete genome sequence of Streptococcus thermophilus DGCC 7710. S. thermophilus is widely used in industrial dairy production.

Development of microsatellites from Fothergilla ×intermedia (Hamamelidaceae) and cross transfer to four other genera within Hamamelidaceae.

PREMISE OF THE STUDY: We developed microsatellites from Fothergilla ×intermedia to establish loci capable of distinguishing species and cultivars, and to assess genetic diversity for use by ornamental breeders and to transfer within Hamamelidaceae. METHODS AND RESULTS: We sequenced a small insert genomic library enriched for microsatellites to develop 12 polymorphic microsatellite loci. The number of alleles detected ranged from four to 15 across five genera within Hamamelidaceae. Shannon's information index ranged from 0.07 to 0.14. CONCLUSIONS: These microsatellite loci provide a set of markers to evaluate genetic diversity of natural and cultivated collections and assist ornamental plant breeders for genetic studies of five popular genera of woody ornamental plants.