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We previously identified specific proteins associated with ethanol stress response in a Lactobacillus buchneri strain capable of growing in 10% ethanol. In the current study, the exceptional roles of ethanol responsive genes are examined to determine if they can increase ethanol tolerance in E. coli host cells. The recombinant strains carrying ethanol responsive genes were subjected to growth analyses in media with and without 4% ethanol. Among the expression of these genes and growth analyses of the recombinant strains in ethanol, six genes Lbuc_0522 (NADPH-dependent methylglyoxal reductase), Lbuc_0354 (succinate semialdehyde dehydrogenase), Lbuc_1211(threonyl_tRNA synthetase), Lbuc_2051 (nitroreductase), Lbuc_0707 (branched chain amino acid aminotransferase) and Lbuc_1852 (proline-specific peptidase) conferred host cells tolerance to 4% ethanol. Six genes Lbuc_1523 (phage major capsid protein, HK 97 family), Lbuc_1319 (phosphoglycerate kinase), Lbuc_0787 encoding fumarylacetoacetate hydrolase, Lbuc_1219 encoding UDP-N-acetylmuramate-L-alanine ligase, Lbuc_0466 encoding ornithine carbamoyltransferase and Lbuc_0858 encoding glycine hydroxymethyltransferase showed no impact on growth in media with 4% ethanol with IPTG induction when compared with E. coli carrying control pET28b plasmid. The expression of two genes Lbuc_1557 (S-layer glycoprotein) and Lbuc_2157 (6-phosphogluconate dehydrogenase) resulted ethanol sensitivity phenotype.

α-Glucans that were enzymatically synthesized from sucrose using glucansucrase cloned from Leuconostoc mesenteroides NRRL B-1118 were found to have a glass transition temperature of approximately 80 °C. Using high-pressure homogenization (~70 MPa), the α-glucans were converted into nanoparticles of ~120 nm in diameter with a surface potential of ~-3 mV. Fluorescence measurements using 1,6-diphenyl-1,3,5-hexatriene (DPH) indicate that the α-glucan nanoparticles have a hydrophobic core that remains intact from 10 to 85 °C. α-Glucan nanoparticles were found to be stable for over 220 days and able to form at three pH levels. Accelerated exposure measurements demonstrated that the α-glucan nanoparticles can endure exposure to elevated temperatures up to 60 °C for 6 h intervals.

Lactobacillus buchneri and Oenococcus oeni are two unique ethanol-tolerant Gram-positive bacteria species. Genome comparison analyses revealed that L. buchneri and O. oeni possess a pntAB locus that was absent in almost all other lactic acid bacteria (LAB) genomes. Our hypothesis is that the pntAB locus contributes to the ethanol tolerance trait of these two distinct ethanol-tolerant organisms. The pntAB locus, consisting of the pntA and pntB genes, codes for NADP(H) transhydrogenase subunits. This membrane-bound transhydrogenase catalyzes the reduction of NADP+ and is known as an important enzyme in maintaining cellular redox balance. In this study, the transhydrogenase operon from L. buchneri NRRL B-30929 and O. oeni PSU-1 were cloned and analyzed. The LbpntB shared 71.0% identity with the O. oeni (OopntB). The entire pntAB locus was expressed in Lactococcus lactis ssp. lactis IL1403 resulting in an increased tolerance to ethanol (6%), butanol (1.8%) and isopropanol (1.8%) when compared to the control strain. However, the recombinant E. coli cells carrying the entire pntAB locus did not show any improved ethanol tolerance. Independent expression of OopntB and LbpntB in recombinant E. coli BL21(DE3)pLysS host demonstrated higher tolerance to ethanol when compared with a control E. coli BL21(DE3)pLysS strain carrying pET28b vector. Ethanol tolerance comparison of E. coli strains carrying LbpntB and OopntB showed that LbpntB conferred higher ethanol tolerance (4.5%) and resulted in greater biomass, while the OopntB conferred lower ethanol tolerance (4.0%) resulted lower biomass. Therefore, the pntB gene from L. buchneri is a better choice in generating higher ethanol tolerance. This is the first study to uncover the role of pntAB locus on ethanol tolerance.

A wide number of Lactic Acid Bacteria (LAB) species produce α-glucans with their ability to synthesize glucansucrases (GS) which use sucrose as substrate for the glucan production. Recently another group of enzymes in LAB gained special interest for their ability to produce α-glucans targeting the substrates containing α1-4-linkages and synthesizing new (α1-6) or (α1-3)-linkages as α‑glucanotransferases. In this study, a putative 4,6‑α‑glucanotransferase (GTFB) from sourdough isolate Lactobacillus reuteri E81 was identified and expressed in Escherichia coli. The biochemical characterization of the GTFB-E81 confirmed its function as it cleaved the α1-4-linkages in different substrates and produced new gluco-oligomers/polymers containing α1-6 linkages together with the α1-4-linkages detected by NMR analysis. GTFB-E81 produced malto-oligosaccharides targeting maltose and maltoheptaose as substrates with up to DP 8 detected by TLC and ESI-MS/MS analysis. The functional roles of these malto-oligosaccharides were determined by testing their immune-modulatory functions in HT29 cells and they triggered the production of anti-inflammatory 1L-4 and pro-inflammatory IL-12 cytokines., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The yajC gene (Lbuc_0921) from Lactobacillus buchneri NRRL B-30929 was identified from previous proteomics analyses in response to ethanol treatment. The YajC protein expression was increased by 15-fold in response to 10 % ethanol vs 0 % ethanol. The yajC gene encodes the smaller subunit of the preprotein translocase complex, which interacts with membrane protein SecD and SecF to coordinate protein transport and secretion across cytoplasmic membrane in Escherichia coli. The YajC protein was linked to sensitivity to growth temperatures in E. coli, involved in translocation of virulence factors during Listeria infection, and stimulating a T cell-mediated response of Brucella abortus. In this study, the L. buchneri yajC gene was over-expressed in E. coli. The strain carrying pET28byajC that produces YajC after isopropyl β-D-1-thiogalactopyranoside induction showed tolerance to 4 % ethanol in growth media, compared to the control carrying pET28b. This is the first report linking YajC to ethanol stress and tolerance.

The production of microbial biomass in liquid media often represents an indispensable step in the research and development of bacterial and fungal strains. Costs of commercially prepared nutrient media or purified media components, however, can represent a significant hurdle to conducting research in locations where obtaining these products is difficult. A less expensive option for providing components essential to microbial growth in liquid culture is the use of extracts of fresh or dried plant products obtained by using hot water extraction techniques. A total of 13 plant extract-based media were prepared from a variety of plant fruits, pods or seeds of plant species including Allium cepa (red onion bulb), Phaseolus vulgaris (green bean pods), and Lens culinaris (lentil seeds). In shake flask tests, cell production by potato dry rot antagonist Pseudomonas fluorescens P22Y05 in plant extract-based media was generally statistically indistinguishable from that in commercially produced tryptic soy broth and nutrient broth as measured by optical density and colony forming units/ml produced (P ≤ 0.05, Fisher's protected LSD). The efficacy of biomass produced in the best plant extract-based media or commercial media was equivalent in reducing Fusarium dry rot by 50-96% compared to controls. In studies using a high-throughput microbioreactor, logarithmic growth of P22Y05 in plant extract-based media initiated in 3-5 h in most cases but specific growth rate and the time of maximum OD varied as did the maximum pH obtained in media. Nutrient analysis of selected media before and after cell growth indicated that nitrogen in the form of NH4 accumulated in culture supernatants, possibly due to unbalanced growth conditions brought on by a scarcity of simple sugars in the media tested. The potential of plant extract-based media to economically produce biomass of microbes active in reducing plant disease is considerable and deserves further research.

Breeding of maize, Zea mays, has improved insect resistance, but the genetic and biochemical basis of many of these improvements is unknown. Maize oligonucleotide microarrays were utilized to identify differentially expressed genes in leaves of three maize inbreds, parents Oh40B and W8 and progeny Oh43, developed in the 1940s. Oh43 had enhanced leaf resistance to corn earworm larvae, Helicoverpa zea, and fall armyworm larvae, Spodoptera frugiperda, compared to one or both parents. Among ca. 100 significantly differentially expressed genes, expression of a Bowman-Birk trypsin inhibitor (BBI) gene was at least ca. 8-fold higher in Oh43 than in either parent. The Oh43 BBI gene was expressed as a recombinant protein. Purified BBI inhibited trypsin and the growth of fall armyworm larvae when added to insect diet. These experiments indicate that comparative gene expression analysis combined with insect resistance measurements of early inbreds can identify previously unrecognized resistance genes.

Rhizopus oryzae is the primary cause of mucormycosis, an emerging, life-threatening infection characterized by rapid angioinvasive growth with an overall mortality rate that exceeds 50%. As a representative of the paraphyletic basal group of the fungal kingdom called "zygomycetes," R. oryzae is also used as a model to study fungal evolution. Here we report the genome sequence of R. oryzae strain 99-880, isolated from a fatal case of mucormycosis. The highly repetitive 45.3 Mb genome assembly contains abundant transposable elements (TEs), comprising approximately 20% of the genome. We predicted 13,895 protein-coding genes not overlapping TEs, many of which are paralogous gene pairs. The order and genomic arrangement of the duplicated gene pairs and their common phylogenetic origin provide evidence for an ancestral whole-genome duplication (WGD) event. The WGD resulted in the duplication of nearly all subunits of the protein complexes associated with respiratory electron transport chains, the V-ATPase, and the ubiquitin-proteasome systems. The WGD, together with recent gene duplications, resulted in the expansion of multiple gene families related to cell growth and signal transduction, as well as secreted aspartic protease and subtilase protein families, which are known fungal virulence factors. The duplication of the ergosterol biosynthetic pathway, especially the major azole target, lanosterol 14alpha-demethylase (ERG11), could contribute to the variable responses of R. oryzae to different azole drugs, including voriconazole and posaconazole. Expanded families of cell-wall synthesis enzymes, essential for fungal cell integrity but absent in mammalian hosts, reveal potential targets for novel and R. oryzae-specific diagnostic and therapeutic treatments., Competing Interests: The authors have declared that no competing interests exist.

Rhizopus oryzae is used for industrial production of lactic acid, yet little is known about the genetics of this fungus. In this study I cloned two genes, ldhA and ldhB, which code for NAD(+)-dependent L-lactate dehydrogenases (LDH) (EC 1.1.1.27), from a lactic acid-producing strain of R. oryzae. These genes are similar to each other and exhibit more than 90% nucleotide sequence identity and they contain no introns. This is the first description of ldh genes in a fungus, and sequence comparisons revealed that these genes are distinct from previously isolated prokaryotic and eukaryotic ldh genes. Protein sequencing of the LDH isolated from R. oryzae during lactic acid production confirmed that ldhA codes for a 36-kDa protein that converts pyruvate to lactate. Production of LdhA was greatest when glucose was the carbon source, followed by xylose and trehalose; all of these sugars could be fermented to lactic acid. Transcripts from ldhB were not detected when R. oryzae was grown on any of these sugars but were present when R. oryzae was grown on glycerol, ethanol, and lactate. I hypothesize that ldhB encodes a second NAD(+)-dependent LDH that is capable of converting L-lactate to pyruvate and is produced by cultures grown on these nonfermentable substrates. Both ldhA and ldhB restored fermentative growth to Escherichia coli (ldhA pfl) mutants so that they grew anaerobically and produced lactic acid.

A genomic DNA library prepared from a benomyl resistant strain of Aspergillus parasiticus was screened with a Neurospora crassa beta-tubulin gene probe. A unique A. parasiticus genomic DNA fragment, thought to carry a mutant beta-tubulin gene (benr), was isolated. Two plasmids, pYT1 and pYTPYRG, carrying the putative benr gene or benr plus a second selectable marker (pyrG), respectively, were used to transform a benomyl sensitive strain of A. parasiticus (CS10) to determine if benr conferred benomyl resistance (BenR). BenR colonies were obtained with pYTPYRG, pYT1 or pYT1 cotransformed with pPG3J which carries a functional pyrG gene. No BenR colonies were obtained without added DNA or with pPG3J only (controls). Southern hybridization analysis of BenR and BenS transformants suggested that plasmid integration occurred most frequently at the chromosomal bens locus, however evidence for gene conversion and heterologous recombination was also observed. The predicted amino acid sequence of benr displayed a high degree of identity (> 93%) with other fungal beta-tubulin genes which confer benomyl resistance. Sequence analysis together with the genetic data suggested that benr encodes a functional mutant beta-tubulin.

The fermentation of cellobiose is a rare trait among yeasts. Of the 308 yeast species that utilize cellobiose aerobically, only 12 species ferment it, and only 2 species, Candida molischiana and Candida wickerhamii, also ferment cellodextrins. Candida molischiana produced beta-glucosidase activity on all carbon sources tested, except glucose, mannose, and fructose. When these sugars were added to cultures growing on cellobiose, the synthesis of beta-glucosidase ceased. However, the total amount of enzyme activity remained constant, indicating that the C. molischiana beta-glucosidase is catabolite repressed and not catabolite inactivated. When grown in medium initially containing glucose plus xylose, cellobiose, maltose, mannitol, or glucitol, C. molischiana preferentially utilized glucose and produced little beta-glucosidase activity until glucose was nearly depleted from the medium. When grown in medium containing cellobiose plus either fructose or mannose, the yeast preferentially utilized the monosaccharides and produced little beta-glucosidase activity. Candida molischiana produced beta-glucosidase and co-utilized cellobiose and xylose, maltose, or trehalose. Glucose and fructose, mannose, or trehalose were co-utilized; however, no beta-glucosidase activity was detected. Thus, the order of substrate preference groups appeared to be (glucose, trehalose, fructose, mannose) > (cellobiose, maltose, xylose) > (mannitol, glucitol).

Spatial knowledge was evaluated in sighted and congenitally blind children using a large-scale four-location navigation task adapted from the work of Landau, Spelke, and Gleitman (1984). From video records we coded the exact path taken and determined accuracy of initial turn, closest position, and final position, relative to target location. We then computed a score to index the efficiency of the path taken. For the sighted sample, after the navigation task, children constructed a tactile map of the test space without the aid of vision and, following removal of the blindfold, drew from memory the spatial layout of the test space. Performance on the navigation and mapping tasks consistently indicated increasing cognitive mapping skills with age in sighted children. Blind children performed comparably to the sighted on all measures except accuracy at final position, for which their performance was worse than that of the sighted. Analysis of the directness of novel paths and other measures taken suggest caution in ascribing well developed Euclidean coding skills to very young children. Results are discussed in light of Landau et al.'s (1984) conclusions.

A cosmid library was constructed by inserting genomic DNA isolated from a wild-type aflatoxin-producing strain of Aspergillus parasiticus (SU-1) into a cosmid vector containing an homologous nitrate reductase (niaD) gene as a selectable marker. One cosmid was isolated which complemented an aflatoxin-deficient, nitrate-nonutilizing mutant strain, A. parasiticus B62 (nor-1, niaD), to aflatoxin production. Deletion and complementation analyses showed that a 1.7 kb BglII-SphI DNA fragment isolated from this cosmid was responsible for renewed aflatoxin production. Northern hybridization analyses revealed that the major RNA transcribed from this DNA fragment was 1.4 kilonucleotides in size. Genetic complementation proved to be a useful strategy for cloning a gene associated with aflatoxin biosynthesis in A. parasiticus.