Identification of rfk-1, a Meiotic Driver Undergoing RNA Editing in Neurospora.

Sk-2 is a meiotic drive element that was discovered in wild populations of Neurospora fungi over 40 years ago. While early studies quickly determined that Sk-2 transmits itself through sexual reproduction in a biased manner via spore killing, the genetic factors responsible for this phenomenon have remained mostly unknown. Here, we identify and characterize rfk-1, a gene required for Sk-2-based spore killing. The rfk-1 gene contains four exons, three introns, and two stop codons, the first of which undergoes RNA editing to a tryptophan codon during sexual development. Translation of an unedited rfk-1 transcript in vegetative tissue is expected to produce a 102-amino acid protein, whereas translation of an edited rfk-1 transcript in sexual tissue is expected to produce a protein with 130 amino acids. These findings indicate that unedited and edited rfk-1 transcripts exist and that these transcripts could have different roles with respect to the mechanism of meiotic drive by spore killing. Regardless of RNA editing, spore killing only succeeds if rfk-1 transcripts avoid silencing caused by a genome defense process called meiotic silencing by unpaired DNA (MSUD). We show that rfk-1's MSUD avoidance mechanism is linked to the genomic landscape surrounding the rfk-1 gene, which is located near the Sk-2 border on the right arm of chromosome III. In addition to demonstrating that the location of rfk-1 is critical to spore-killing success, our results add to accumulating evidence that MSUD helps protect Neurospora genomes from complex meiotic drive elements.

Convergent evolution of complex genomic rearrangements in two fungal meiotic drive elements.

Meiotic drive is widespread in nature. The conflict it generates is expected to be an important motor for evolutionary change and innovation. In this study, we investigated the genomic consequences of two large multi-gene meiotic drive elements, Sk-2 and Sk-3, found in the filamentous ascomycete Neurospora intermedia. Using long-read sequencing, we generated the first complete and well-annotated genome assemblies of large, highly diverged, non-recombining regions associated with meiotic drive elements. Phylogenetic analysis shows that, even though Sk-2 and Sk-3 are located in the same chromosomal region, they do not form sister clades, suggesting independent origins or at least a long evolutionary separation. We conclude that they have in a convergent manner accumulated similar patterns of tandem inversions and dense repeat clusters, presumably in response to similar needs to create linkage between genes causing drive and resistance.

An RNA Recognition Motif-Containing Protein Functions in Meiotic Silencing by Unpaired DNA.

Meiotic silencing by unpaired DNA (MSUD) is a biological process that searches pairs of homologous chromosomes (homologs) for segments of DNA that are unpaired. Genes found within unpaired segments are silenced for the duration of meiosis. In this report, we describe the identification and characterization of Neurospora crassa sad-7, a gene that encodes a protein with an RNA recognition motif (RRM). Orthologs of sad-7 are found in a wide range of ascomycete fungi. In N. crassa, sad-7 is required for a fully efficient MSUD response to unpaired genes. Additionally, at least one parent must have a functional sad-7 allele for a cross to produce ascospores. Although sad-7-null crosses are barren, sad-7Δ strains grow at a wild-type (wt) rate and appear normal under vegetative growth conditions. With respect to expression, sad-7 is transcribed at baseline levels in early vegetative cultures, at slightly higher levels in mating-competent cultures, and is at its highest level during mating. These findings suggest that SAD-7 is specific to mating-competent and sexual cultures. Although the role of SAD-7 in MSUD remains elusive, green fluorescent protein (GFP)-based tagging studies place SAD-7 within nuclei, perinuclear regions, and cytoplasmic foci of meiotic cells. This localization pattern is unique among known MSUD proteins and raises the possibility that SAD-7 coordinates nuclear, perinuclear, and cytoplasmic aspects of MSUD.

Polyols, not sugars, determine the structural diversity of anti-streptococcal liamocins produced by Aureobasidium pullulans strain NRRL 50380.

Liamocins are polyol lipids produced by the fungus Aureobasidium pullulans, and have selective antibacterial activity against Streptococcus species. Liamocins produced by A. pullulans strain NRRL 50380 on sucrose medium have a d-mannitol head group ester-linked to 3,5-dihydroxydecanoate acyl chains, three or four of which are joined together by 1,5-polyester bonds (liamocins Man-A1 and Man-B1), and similar 3'-O-acetylated analogs (Man-A2 and Man-B2). However, other types of liamocins are produced depending on the choice of strain and growth conditions. In the current study, growth on different polyols, but not sugars, resulted in considerable structural variation, including liamocins with d-galactitol (dulcitol), d-sorbitol (glucitol), d- and l-arabitol, d-xylitol, l-threitol and glycerol head groups. The head groups of liamocins produced on arabitol were shown to be entirely composed of d-arabitol. These liamocin variants were structurally characterized by NMR and MS, and tested for antibacterial activity. The new liamocin variants also had selective activity against Streptococcus. Liamocin structural variants are novel antibacterials against Streptococcus sp. that merit further investigation.

Production of anti-streptococcal liamocins from agricultural biomass by Aureobasidium pullulans.

Liamocins are unique heavier-than-water "oils" produced by certain strains of the fungus Aureobasidium pullulans. Liamocins have antibacterial activity with specificity for Streptococcus sp. Previous studies reported that liamocin yields were highest from strains of A. pullulans belonging to phylogenetic clades 8, 9, and 11, cultured on medium containing sucrose. In this study, 27 strains from these clades were examined for the first time for production of liamocins from agricultural biomass substrates. Liamocin yields were highest from strains in phylogenetic clade 11, and yields were higher from cultures grown on sucrose than from those grown on pretreated wheat straw. However, when supplementary enzymes (cellulase, ß-glucosidase, and xylanase) were added, liamocin production on pretreated wheat straw was equivalent to that on sucrose. Liamocins produced from wheat straw were free of the melanin contamination common in sucrose-grown cultures. Furthermore, MALDI-TOF MS analysis showed that liamocins produced from wheat straw were under-acetylated, resulting in higher proportions of the mannitol A1 and B1 species of liamocin, the latter of which has the highest biological activity against Streptococcus sp.

Production of high activity Aspergillus niger BCC4525 ß-mannanase in Pichia pastoris and its application for mannooligosaccharides production from biomass hydrolysis.

A cDNA encoding ß-mannanase was cloned from Aspergillus niger BCC4525 and expressed in Pichia pastoris KM71. The secreted enzyme hydrolyzed locust bean gum substrate with very high activity (1625 U/mL) and a relatively high kcat/Km (461 mg-1 s-1 mL). The enzyme is thermophilic and thermostable with an optimal temperature of 70 °C and 40% retention of endo-ß-1,4-mannanase activity after preincubation at 70 °C. In addition, the enzyme exhibited broad pH stability with an optimal pH of 5.5. The recombinant enzyme hydrolyzes low-cost biomass, including palm kernel meal (PKM) and copra meal, to produce mannooligosaccharides, which is used as prebiotics to promote the growth of beneficial microflora in animals. An in vitro digestibility test simulating the gastrointestinal tract system of broilers suggested that the recombinant ß-mannanase could effectively liberate reducing sugars from PKM-containing diet. These characteristics render this enzyme suitable for utilization as a feed additive to improve animal performance.

Phylogenetic classification of Aureobasidium pullulans strains for production of feruloyl esterase.

OBJECTIVE: The objective was to phylogenetically classify diverse strains of Aureobasidium pullulans and determine their production of feruloyl esterase. RESULTS: Seventeen strains from the A. pullulans literature were phylogenetically classified. Phenotypic traits of color variation and endo-ß-1,4-xylanase overproduction were associated with phylogenetic clade 10 and particularly clade 8. Literature strains used for pullulan production all belonged to clade 7. These strains and 36 previously classified strains were tested for feruloyl esterase production, which was found to be associated with phylogenetic clades 4, 11, and particularly clade 8. Clade 8 strains NRRL 58552 and NRRL 62041 produced the highest levels of feruloyl esterase among strains tested. CONCLUSIONS: Production of both xylanase and feruloyl esterase are associated with A. pullulans strains in phylogenetic clade 8, which is thus a promising source of enzymes with potential biotechnological applications.

Production of novel types of antibacterial liamocins by diverse strains of Aureobasidium pullulans grown on different culture media.

OBJECTIVES: To compare production of antibacterial liamocins (polyol lipids) by diverse strains of Aureobasidium pullulans grown on different culture media. RESULTS: Liamocins produced by strains of A. pullulans have potential agricultural and pharmaceutical applications as antibacterials with specificity against Streptococcus spp. Six strains of A. pullulans were characterized for liamocin production on four different culture media. The choice of strain and culture medium affected growth, liamocin yields, and production of contaminating pigments. Best growth and highest liamocin yields were obtained using A. pullulans strain NRRL 50384 grown on a sucrose basal medium. Unexpectedly, the choice of strain and culture medium also affected the structure of liamocins produced, providing novel types of liamocins. Liamocins varied not only in the ratios of trimer and tetramer polyester tail groups, but also in the nature of the polyol headgroup, which could include mannitol, arabitol, or glycerol. CONCLUSIONS: The ability to conveniently produce novel types of liamocins in good yields will provide novel antibacterials for applied uses, and facilitate structure-function studies on the mechanism of antibacterial activity.

Inhibitors of biofilm formation by biofuel fermentation contaminants.

Biofuel fermentation contaminants such as Lactobacillus sp. may persist in production facilities by forming recalcitrant biofilms. In this study, biofilm-forming strains of Lactobacillus brevis, Lactobacillus fermentum, and Lactobacillus plantarum were isolated and characterized from a dry-grind fuel ethanol plant. A variety of potential biofilm inhibitors were tested, including microbial polysaccharides, commercial enzymes, ferric ammonium citrate, liamocins, phage endolysin, xylitol, and culture supernatants from Bacillus sp. A commercial enzyme mixture (Novozyme 188) and culture supernatants from Bacillus subtilis strains ALT3A and RPT-82412 were identified as the most promising biofilm inhibitors. In biofilm flow cells, these inhibitors reduced the density of viable biofilm cells by 0.8-0.9 log cfu/cm(2). Unlike B. subtilis strain RPT-82412, B. subtilis strain ALT3A and Novozyme 188 did not inhibit planktonic growth of Lactobacillus sp. MALDI-TOF mass spectra showed the production of surfactin-like molecules by both B. subtilis strains, and the coproduction of iturin-like molecules by strain RPT-82412.

Aureobasidium pullulans as a source of liamocins (heavy oils) with anticancer activity.

Liamocins are structurally unique, heavier-than-water "oils" produced by certain strains of Aureobasidium pullulans. The aim of the current study is to identify new sources of liamocins and evaluate their potential as anticancer agents. Nine strains of A. pullulans from phylogenetic clades 8, 9, and 11 were examined for the first time for production of liamocins. Strains in these clades have only been isolated from tropical environments, and all strains tested here were from various locations in Thailand. Strains RSU 9, RSU 21, and RSU 29, all from clade 11, produced from 7.0 to 8.6 g liamocins/l from medium containing 5 % sucrose. These are the highest yields of liamocins that we have found thus far. These strains also produced from 9.4 to 17 g pullulan/l. The structural identity of liamocins was confirmed by matrix-assisted laser desorption/ionization mass spectrometry; differential spectra were obtained in which the dominant ion was either at about m/z 805.5 or m/z 949.6, consistent with the structure of liamocins. Liamocins from A. pullulans strains RSU 9 and RSU 21 inhibited two human breast cancer cell lines and a human cervical cancer cell line (IC50 values of 32.2 ± 1.4 to 63.1 ± 2.4 µg liamocins/ml) but were not toxic to a normal cell line. Liamocins weakly inhibited a strain of Enterococcus faecalis, but did not inhibit strains of Lactobacillus fermentum, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Thus, A. pullulans phylogenetic clade 11 is a promising source of liamocins, and these compounds merit further examination as potential anticancer agents.

Lipase production by diverse phylogenetic clades of Aureobasidium pullulans.

Thirty-nine strains representing 12 diverse phylogenetic clades of Aureobasidium pullulans were surveyed for lipase production using a quantitative assay. Strains in clades 4 and 10 produced 0.2-0.3 U lipase/ml, while color variant strain NRRL Y-2311-1 in clade 8 produced 0.54 U lipase/ml. Strains in clade 9, which exhibit a dark olivaceous pigment, produced the highest levels of lipase, with strain NRRL 62034 yielding 0.57 U lipase/ml. By comparison, Candida cylindracea strain NRRL Y-17506 produced 0.05 U lipase/ml under identical conditions. A. pullulans strain NRRL 62034 reached maximal lipase levels in 5 days on lipase induction medium, while A. pullulans strain NRRL Y-2311-1 and strains in clades 4 and 10 were highest after 6 days. A. pullulans strain NRRL Y-2311-1 and strains in clade 9 produced two extracellular proteins in common, at >50 and <37 kDa.

Laccases from Aureobasidium pullulans.

Laccases are polyphenol oxidases (EC 1.10.3.2) that have numerous industrial and bioremediation applications. Laccases are well known as lignin-degrading enzymes, but these enzymes can play numerous other roles in fungi. In this study, 41 strains of the fungus Aureobasidium pullulans were examined for laccase production. Enzymes from A. pullulans were distinct from those from lignin-degrading fungi and associated with pigment production. Laccases from strains in phylogenetic clade 5, which produced a dark vinaceous pigment, exhibited a temperature optimum of 50-60°C and were stable for an hour at 50°C, unlike enzymes from the lignin-degrading fungi Trametes versicolor and Pycnoporus cinnabarinus. Laccase purified from A. pullulans strain NRRL 50381, a representative of clade 5, was glycosylated but had a molecular weight of 60-70kDa after Endo H treatment. Laccase purified from strain NRRL Y-2568, which produced a dark olivaceous pigment, was also glycosylated, but had a molecular weight of greater than 100kDa after Endo H treatment.

Structural characterization of novel extracellular liamocins (mannitol oils) produced by Aureobasidium pullulans strain NRRL 50380.

Aureobasidium pullulans is a common, ubiquitous fungus, which is used industrially to produce the polysaccharide pullulan. We have previously shown that A. pullulans produces various heavier-than-water oils, first named here as liamocins, that accumulate in fermentations. Here we report the structural characterization of four liamocins, A1, A2, B1, and B2, produced by A. pullulans strain NRRL 50380 using a combination of MALDI-TOF/MS, quadrupole-TOF/MS, isotopic labeling, NMR, GC/MS, and classical carbohydrate analysis. The data showed that the liamocins are composed of a single mannitol headgroup partially O-acylated with three (for liamocin A1 and A2) or four (for liamocin B1 and B2) 3,5-dihydroxydecanoic ester groups. Liamocins A1 and B1 are non-acetylated, whereas A2 and B2 each contain a single 3'-O-acetyl group. Each of these compounds is characterized by pseudomolecular [M+Na](+) ions in the MALDI-TOF/MS spectra at m/z 763.22, 949.35, 805.22, and 991.37, respectively. The 186Da mass difference between A-type and B-type liamocins corresponds to one O-linked 3,5-dihydroxydecanoate group. HMBC NMR showed that one 3,5-dihydroxydecanoate carbonyl group is ester linked to a primary hydroxyl on the mannitol. Other long range (13)C-(1)H couplings across 1,5-ester bridges showed that the 3,5-dihydroxydecanoate groups form 1-5-linked polyester chains, similar in structure to the antibiotic substance exophilin A. Moreover, the MS analysis identified several non-conjugated poly-3,5-dihydroxydecanoate esters as minor components that are tentatively assigned as exophilins A1, A2, B1, and B2. The liamocins, and three of the exophilins, are new, previously unreported structures.

Bacillus spp. produce antibacterial activities against lactic acid bacteria that contaminate fuel ethanol plants.

Lactic acid bacteria (LAB) frequently contaminate commercial fuel ethanol fermentations, reducing yields and decreasing profitability of biofuel production. Microorganisms from environmental sources in different geographic regions of Thailand were tested for antibacterial activity against LAB. Four bacterial strains, designated as ALT3A, ALT3B, ALT17, and MR1, produced inhibitory effects on growth of LAB. Sequencing of rRNA identified these strains as species of Bacillus subtilis (ALT3A and ALT3B) and B. cereus (ALT17 and MR1). Cell mass from colonies and agar samples from inhibition zones were analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The spectra of ALT3A and ALT3B showed a strong signal at m/z 1,060, similar in mass to the surfactin family of antimicrobial lipopeptides. ALT3A and ALT3B were analyzed by zymogram analysis using SDS-PAGE gels placed on agar plates inoculated with LAB. Cell lysates possessed an inhibitory protein of less than 10 kDa, consistent with the production of an antibacterial lipopeptide. Mass spectra of ALT17 and MR1 had notable signals at m/z 908 and 930 in the whole cell extracts and at m/z 687 in agar, but these masses do not correlate with those of previously reported antibacterial lipopeptides, and no antibacterial activity was detected by zymogram. The antibacterial activities produced by these strains may have application in the fuel ethanol industry as an alternative to antibiotics for prevention and control of bacterial contamination.

Production of poly(ß-L-malic acid) (PMA) from agricultural biomass substrates by Aureobasidium pullulans.

For the first time the production of poly(ß-L -malic acid) (PMA) has been achieved using agricultural biomass substrates by the yeast-like fungus Aureobasidium pullulans. Strains NRRL Y-2311-1, NRRL 50382, NRRL 50383, and NRRL 50384, representing diverse isolation sources and phylogenetic clades, produced PMA from alkaline H(2)O(2)-pretreated corn fiber and wheat straw as sole carbon sources. Pretreated wheat straw was better than pretreated corn fiber, and strain NRRL 50383 gave the highest overall yields of PMA. The addition of CaCO(3) plus supplementary hydrolytic enzymes enhanced PMA production. Four basal media were compared for PMA production, and the best was found to be a N-limited pullulan production medium (PM). In this medium, PMA production took place during growth limitation. Under optimal conditions, strain NRRL 50383 produced more than 20 g PMA/l from 5 % (w/v) pretreated wheat straw in PM with 3 % (w/v) CaCO(3) and supplementary enzymes.

Aureobasidium thailandense sp. nov. isolated from leaves and wooden surfaces.

Aureobasidium thailandense sp. nov. is described from cultures of material collected on leaves and wooden surfaces in Thailand and the type isolate is NRRL 58539(T). Phylogenetically it is distinct from other species of the genus Aureobasidium. Phenotypically it is distinguished by its cardinal growth temperatures, salt tolerance and production of reddish brown hyphal pigmentation in PDA cultures, but micro-morphologically it is not clearly distinguishable from Aureobasidium pullulans. Unlike A. pullulans, A. thailandense sp. nov. produces a non-pullulan extracellular polysaccharide whose characteristics are unknown. The two known isolates of A. thailandense sp. nov. possess an approx. 500 bp type I intron in the 18S rRNA gene that is present in ITS amplifications using primers ITS4 and ITS5. A. pullulans isolates uniformly lack this intron.

Poly(ß-L-malic acid) production by diverse phylogenetic clades of Aureobasidium pullulans.

Poly(ß-L-malic acid) (PMA) is a natural biopolyester that has pharmaceutical applications and other potential uses. In this study, we examined PMA production by 56 strains of the fungus Aureobasidium pullulans representing genetically diverse phylogenetic clades. Thirty-six strains were isolated from various locations in Iceland and Thailand. All strains from Iceland belonged to a newly recognized clade 13, while strains from Thailand were distributed among 8 other clades, including a novel clade 14. Thirty of these isolates, along with 26 previously described strains, were examined for PMA production in medium containing 5% glucose. Most strains produced at least 4 g PMA/L, and several strains in clades 9, 11, and 13 made 9-11 g PMA/L. Strains also produced both pullulan and heavy oil, but PMA isolated by differential precipitation in ethanol exhibited up to 72% purity with no more than 12% contamination by pullulan. The molecular weight of PMA from A. pullulans ranged from 5.1 to 7.9 kDa. Results indicate that certain genetic groups of A. pullulans are promising for the production of PMA.

Heavy oils produced by Aureobasidium pullulans.

From a survey of more than 50 diverse strains of Aureobasidium pullulans, 21 produced extracellular heavy oils. Most oil producers fell into phylogenetic clades 8, 9, and 11. Oil colors ranged from bright yellow to malachite. More than half of the strains produced oil that was fluorescent. In medium containing 5% (w/v) sucrose, oil yields ranged from 0.5 to 6 g oil/l. Strain CU 43 reached stationary growth phase at day 4 while oil yields were maximal at day 6. CU 43 produced bright yellow, highly fluorescent oil that also was visible as intracellular droplets under fluorescent microscopy. Oil was surface active, suggesting that it functions as a biosurfactant. Oil from two strains (CU 43 and NRRL Y-12974) differentially inhibited mammalian cancer cell lines. MALDI-TOF MS spectra suggested that A. pullulans strains produce a family of related oil structures.

α-Amylase activity during pullulan production and α-amylase gene analyses of Aureobasidium pullulans.

Aureobasidium pullulans is the source of commercially produced pullulan, a high molecular weight polysaccharide that is used in the manufacture of edible films. It has been proposed that α-amylase decreases the molecular weight of pullulan in late cultures. Based on a recent phylogenetic analysis, five representative strains were chosen to study the relationship between α-amylase and pullulan production. In sucrose-grown cultures, pullulan yields increased over time while the molecular weight of pullulan generally decreased. However, no α-amylase activity was detected in these cultures. Low levels of α-amylase were present in starch-grown culture, but pullulan analysis was complicated by residual starch. To facilitate further studies on the role of α-amylase in the reduction of pullulan molecular weight, the α-amylase gene from A. pullulans NRRL Y-12974 was cloned and characterized. The coding region of the complete α-amylase gene contains 2,247 bp, including 7 introns and 8 exons. The putative mRNA was 1,878 bp long, encoding an α-amylase of 625 amino acid residues. Southern blot analysis indicated that there was only one copy of this gene in the genome. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that the gene was transcribed in both sucrose- and starch-grown cultures. It is possible that very low levels of α-amylase attack the minor maltotetraose subunits of pullulan and cause the reduction of molecular weight.