Natural allelic variations ofSaccharomyces cerevisiaeimpact stuck fermentation due to the combined effect of ethanol and temperature; a QTL-mapping study

AbstractBackgroundFermentation completion is a major prerequisite in many industrial processes involving the bakery yeastSaccharomyces cerevisiae.Stuck fermentations can be due to the combination of many environmental stresses. Among them high temperature and ethanol content are particularly deleterious especially in bioethanol and red wine production. Although the genetic causes of temperature and/or ethanol tolerance were widely investigated in laboratory conditions, few studies investigated natural genetic variations related to stuck fermentations in high gravity matrixes.ResultsIn this study, three QTLs linked to stuck fermentation in winemaking conditions were identified by using a selective genotyping strategy carried out on a backcrossed population. The precision of mapping allows the identification of two causative genesVHS1andOYE2characterized by stop-codon insertion. The phenotypic effect of these allelic variations was validated by Reciprocal Hemyzygous Assay in high gravity fermentations (>240 g/L of sugar) carried out at high temperatures (>28°C). Phenotypes impacted were related to the late stage of alcoholic fermentation during the stationary growth phase of yeast.ConclusionsThe genes identified are related to molecular functions such as Programed Cell Death, ROS metabolism and respire-fermentative switch and were never related to fermentation efficiency. Our findings open new avenues for better understanding yeast resistance mechanisms involved in high gravity fermentations.

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Overlapping reading frames at the LYS5 locus in the yeast Yarrowia lipolytica

Molecular and Cellular Biology ◽

10.1128/mcb.10.9.4795-4806.1990 ◽

1990 ◽

Vol 10 (9) ◽

pp. 4795-4806

Author(s):

J W Xuan ◽

P Fournier ◽

N Declerck ◽

M Chasles ◽

C Gaillardin

Keyword(s):

Yarrowia Lipolytica ◽

Stop Codon ◽

Open Reading Frames ◽

Yeast Cells ◽

Lacz Gene ◽

Galactosidase Activity ◽

Phenotypic Effect ◽

Frame Fusion ◽

Beta Galactosidase ◽

Reading Frames

Mutants affected at the LYS5 locus of Yarrowia lipolytica lack detectable dehydrogenase (SDH) activity. The LYS5 gene has previously been cloned, and we present here the sequence of the 2.5-kilobase-pair (kb) DNA fragment complementing the lys5 mutation. Two large antiparallel open reading frames (ORF1 and ORF2) were observed, flanked by potential transcription signals. Both ORFs appear to be transcribed, but several lines of evidence suggest that only ORF2 is translated and encodes SDH. (i) The global amino acid compositions of Saccharomyces cerevisiae SDH and of the putative ORF2 product are similar and that of ORF1 is dissimilar. (ii) An in-frame translational fusion of ORF2 with the Escherichia coli lacZ gene was introduced into yeast cells and resulted in a beta-galactosidase activity regulated similarly to SDH; no beta-galactosidase activity was obtained with an in-frame fusion of ORF1 with lacZ. (iii) The introduction of a stop codon at the beginning of ORF2 prevented SDH expression in yeast cells, whereas no phenotypic effect was observed when ORF1 translation was blocked.

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Overlapping reading frames at the LYS5 locus in the yeast Yarrowia lipolytica.

Molecular and Cellular Biology ◽

10.1128/mcb.10.9.4795 ◽

1990 ◽

Vol 10 (9) ◽

pp. 4795-4806 ◽

Cited By ~ 29

Author(s):

J W Xuan ◽

P Fournier ◽

N Declerck ◽

M Chasles ◽

C Gaillardin

Keyword(s):

Yarrowia Lipolytica ◽

Stop Codon ◽

Open Reading Frames ◽

Yeast Cells ◽

Lacz Gene ◽

Galactosidase Activity ◽

Phenotypic Effect ◽

Frame Fusion ◽

Beta Galactosidase ◽

Reading Frames

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In VitroActivities of the Novel Investigational Tetrazoles VT-1161 and VT-1598 Compared to the Triazole Antifungals against Azole-Resistant Strains and Clinical Isolates ofCandida albicans

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00341-19 ◽

2019 ◽

Vol 63 (6) ◽

Cited By ~ 4

Author(s):

Andrew T. Nishimoto ◽

Nathan P. Wiederhold ◽

Stephanie A. Flowers ◽

Qing Zhang ◽

Steven L. Kelly ◽

...

Keyword(s):

Fungal Infections ◽

Stop Codon ◽

Geometric Mean ◽

Premature Stop Codon ◽

Resistance Mechanisms ◽

Clinical Isolates ◽

Azole Resistance ◽

Content Type ◽

Mutant Strains

ABSTRACTThe fungal Cyp51-specific inhibitors VT-1161 and VT-1598 have emerged as promising new therapies to combat fungal infections, includingCandidaspp. To evaluate theirin vitroactivities compared to other azoles, MICs were determined by Clinical and Laboratory Standards Institute (CLSI) method for VT-1161, VT-1598, fluconazole, voriconazole, itraconazole, and posaconazole against 68 C. albicansclinical isolates well characterized for azole resistance mechanisms and mutant strains representing individual azole resistance mechanisms. VT-1161 and VT-1598 demonstrated potent activity (geometric mean MICs ≤0.15 μg/ml) against predominantly fluconazole-resistant (≥8 μg/ml) isolates. However, five of 68 isolates exhibited MICs greater than six dilutions (>2 μg/ml) to both tetrazoles compared to fluconazole-susceptible isolates. Four of these isolates likewise exhibited high MICs beyond the upper limit of the assay for all triazoles tested. A premature stop codon inERG3likely explained the high-level resistance in one isolate. VT-1598 was effective against strains with hyperactive Tac1, Mrr1, and Upc2 transcription factors and against mostERG11mutant strains. VT-1161 MICs were elevated compared to the control strain SC5314 for hyperactive Tac1 strains and two strains with Erg11 substitutions (Y132F and Y132F&K143R) but showed activity against hyperactive Mrr1 and Upc2 strains. While mutations affecting Erg3 activity appear to greatly reduce susceptibility to VT-1161 and VT-1598, the elevated MICs of both tetrazoles for four isolates could not be explained by known azole resistance mechanisms, suggesting the presence of undescribed resistance mechanisms to triazole- and tetrazole-based sterol demethylase inhibitors.

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AMRFinderPlus and the Reference Gene Catalog facilitate examination of the genomic links among antimicrobial resistance, stress response, and virulence

Scientific Reports ◽

10.1038/s41598-021-91456-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Michael Feldgarden ◽

Vyacheslav Brover ◽

Narjol Gonzalez-Escalona ◽

Jonathan G. Frye ◽

Julie Haendiges ◽

...

Keyword(s):

Stress Response ◽

Antimicrobial Resistance ◽

Reference Gene ◽

Virulence Genes ◽

Stop Codon ◽

Point Mutations ◽

Resistance Mechanisms ◽

Gene Content ◽

Gene Catalog ◽

Gene Database

AbstractAntimicrobial resistance (AMR) is a significant public health threat. With the rise of affordable whole genome sequencing, in silico approaches to assessing AMR gene content can be used to detect known resistance mechanisms and potentially identify novel mechanisms. To enable accurate assessment of AMR gene content, as part of a multi-agency collaboration, NCBI developed a comprehensive AMR gene database, the Bacterial Antimicrobial Resistance Reference Gene Database and the AMR gene detection tool AMRFinder. Here, we describe the expansion of the Reference Gene Database, now called the Reference Gene Catalog, to include putative acid, biocide, metal, stress resistance genes, in addition to virulence genes and species-specific point mutations. Genes and point mutations are classified by broad functions, as well as more detailed functions. As we have expanded both the functional repertoire of identified genes and functionality, NCBI released a new version of AMRFinder, known as AMRFinderPlus. This new tool allows users the option to utilize only the core set of AMR elements, or include stress response and virulence genes, too. AMRFinderPlus can detect acquired genes and point mutations in both protein and nucleotide sequence. In addition, the evidence used to identify the gene has been expanded to include whether nucleotide or protein sequence was used, its location in the contig, and presence of an internal stop codon. These database improvements and functional expansions will enable increased precision in identifying AMR genes, linking AMR genotypes and phenotypes, and determining possible relationships between AMR, virulence, and stress response.

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Improved Fermentation Performance of a Lager Yeast after Repair of Its AGT1 Maltose and Maltotriose Transporter Genes

Applied and Environmental Microbiology ◽

10.1128/aem.01558-08 ◽

2009 ◽

Vol 75 (8) ◽

pp. 2333-2345 ◽

Cited By ~ 48

Author(s):

Virve Vidgren ◽

Anne Huuskonen ◽

Hannele Virtanen ◽

Laura Ruohonen ◽

John Londesborough

Keyword(s):

Stop Codon ◽

Premature Stop Codon ◽

High Gravity ◽

Transport Activity ◽

Lager Yeast ◽

Very High Gravity ◽

Maltose Transport ◽

Yeast Strains ◽

Brewer’S Yeasts ◽

Very High

ABSTRACT The use of more concentrated, so-called high-gravity and very-high-gravity (VHG) brewer's worts for the manufacture of beer has economic and environmental advantages. However, many current strains of brewer's yeasts ferment VHG worts slowly and incompletely, leaving undesirably large amounts of maltose and especially maltotriose in the final beers. α-Glucosides are transported into Saccharomyces yeasts by several transporters, including Agt1, which is a good carrier of both maltose and maltotriose. The AGT1 genes of brewer's ale yeast strains encode functional transporters, but the AGT1 genes of the lager strains studied contain a premature stop codon and do not encode functional transporters. In the present work, one or more copies of the AGT1 gene of a lager strain were repaired with DNA sequence from an ale strain and put under the control of a constitutive promoter. Compared to the untransformed strain, the transformants with repaired AGT1 had higher maltose transport activity, especially after growth on glucose (which represses endogenous α-glucoside transporter genes) and higher ratios of maltotriose transport activity to maltose transport activity. They fermented VHG (24° Plato) wort faster and more completely, producing beers containing more ethanol and less residual maltose and maltotriose. The growth and sedimentation behaviors of the transformants were similar to those of the untransformed strain, as were the profiles of yeast-derived volatile aroma compounds in the beers.

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Low Frequency of Ceftazidime-Avibactam Resistance among Enterobacteriaceae Isolates Carrying blaKPC Collected in U.S. Hospitals from 2012 to 2015

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02369-16 ◽

2016 ◽

Vol 61 (3) ◽

Cited By ~ 24

Author(s):

Mariana Castanheira ◽

Rodrigo E. Mendes ◽

Helio S. Sader

Keyword(s):

Efflux Pump ◽

Stop Codon ◽

Premature Stop Codon ◽

Low Frequency ◽

Resistance Mechanisms ◽

Sequencing Analysis ◽

Relative Quantification ◽

Large Collection ◽

Icu Patients ◽

Content Type

ABSTRACT Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae isolates have been increasingly reported worldwide, and therapeutic options to treat infections caused by these organisms are limited. We evaluated the activity of ceftazidime-avibactam and comparators against 456 Enterobacteriaceae isolates carrying bla KPC collected from 79 U.S. hospitals during 2012 to 2015. Overall, ceftazidime-avibactam (MIC50/90, 0.5/2 μg/ml; 99.3% susceptible) and tigecycline (MIC50/90, 0.5/1 μg/ml; 98.9% susceptible at ≤2 μg/ml) were the most active agents. Only 80.5% and 59.0% of isolates were susceptible to colistin and amikacin, respectively. All three isolates (0.7%) displaying resistance to ceftazidime-avibactam (K. pneumoniae; MICs, ≥16 μg/ml) were evaluated using whole-genome sequencing analysis and relative quantification of expression levels of porins and efflux pump. Two isolates carried metallo-β-lactamase genes, bla NDM-1 or bla VIM-4, among other β-lactam resistance mechanisms, and one displayed a premature stop codon in ompK35 and decreased expression of ompK36. Ceftazidime-avibactam was active against 100.0 and 99.3% of isolates carrying bla KPC-3 (n = 221) and bla KPC-2 (n = 145), respectively. Isolates carrying bla KPC were more commonly recovered from pneumonia (n = 155), urinary tract (n = 93), and skin/soft tissue (n = 74) infections. Ceftazidime-avibactam (97.8 to 100.0% susceptible) was consistently active against isolates from all infection sites. K. pneumoniae (83.3% of the collection) susceptibility rates were 99.2% for ceftazidime-avibactam, 98.9% for tigecycline, and 80.1% for colistin. Ceftazidime-avibactam susceptibility did not vary substantially when comparing isolates from intensive care unit (ICU) patients to those from non-ICU patients. Ceftazidime-avibactam was active against this large collection of isolates carrying bla KPC and represents a valuable addition to the armamentarium currently available for the treatment of infections caused by KPC-producing Enterobacteriaceae.

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Targeted Gene Mutation in Phytophthora spp.

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-19-1359 ◽

2006 ◽

Vol 19 (12) ◽

pp. 1359-1367 ◽

Cited By ~ 17

Author(s):

Kurt H. Lamour ◽

Ledare Finley ◽

Oscar Hurtado-Gonzales ◽

Daniel Gobena ◽

Melinda Tierney ◽

...

Keyword(s):

Plant Pathogens ◽

Genomic Library ◽

Stop Codon ◽

Point Mutations ◽

Premature Stop Codon ◽

Phytophthora Sojae ◽

Phenotypic Effect ◽

Phytophthora Spp ◽

Homozygous Mutant ◽

Genes Encoding

The genus Phytophthora belongs to the oomycetes and is composed of plant pathogens. Currently, there are no strategies to mutate specific genes for members of this genus. Whole genome sequences are available or being prepared for Phytophthora sojae, P. ramorum, P. infestans, and P. capsici and the development of molecular biological techniques for functional genomics is encouraged. This article describes the adaptation of the reverse-genetic strategy of targeting induced local lesions in genomes (TILLING) to isolate gene-specific mutants in Phytophthora spp. A genomic library of 2,400 ethylnitrosourea (ENU) mutants of P. sojae was created and screened for induced point mutations in the genes encoding a necrosis-inducing protein (PsojNIP) and a Phytophthora-specific phospholipase D (PsPXTM-PLD). Mutations were detected in single individuals and included silent, missense, and nonsense changes. Homozygous mutant isolates carrying a potentially deleterious missense mutation in PsojNIP and a premature stop codon in PsPXTM-PLD were identified. No phenotypic effect has yet been found for the homozygous mutant of PsojNIP. For those of PsPXTM-PLD, a reduction in growth rate and an appressed mycelial growth was observed. This demonstrates the feasibility of target-selected gene disruption for Phytophthora spp. and adds an important tool for functional genomic investigation.

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Analysis of the Essential Cell Division Gene ftsL ofBacillus subtilis by Mutagenesis and Heterologous Complementation

Journal of Bacteriology ◽

10.1128/jb.182.19.5572-5579.2000 ◽

2000 ◽

Vol 182 (19) ◽

pp. 5572-5579 ◽

Cited By ~ 15

Author(s):

Jörg Sievers ◽

Jeff Errington

Keyword(s):

Cell Division ◽

Leucine Zipper ◽

Stop Codon ◽

Random Mutagenesis ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Missense Mutations ◽

Loss Of Function ◽

Induced Mutations ◽

Phenotypic Effect

ABSTRACT The ftsL gene is required for the initiation of cell division in a broad range of bacteria. Bacillus subtilis ftsL encodes a 13-kDa protein with a membrane-spanning domain near its N terminus. The external C-terminal domain has features of an α-helical leucine zipper, which is likely to be involved in the heterodimerization with another division protein, DivIC. To determine what residues are important for FtsL function, we used both random and site-directed mutagenesis. Unexpectedly, all chemically induced mutations fell into two clear classes, those either weakening the ribosome-binding site or producing a stop codon. It appears that the random mutagenesis was efficient, so many missense mutations must have been generated but with no phenotypic effect. Substitutions affecting hydrophobic residues in the putative coiled-coil domain, introduced by site-directed mutagenesis, also gave no observable phenotype except for insertion of a helix-breaking proline residue, which destroyed FtsL function. ftsL homologues cloned from three diverseBacillus species, Bacillus licheniformis,Bacillus badius, and Bacillus circulans, could complement an ftsL null mutation in B. subtilis, even though up to 66% of the amino acid residues of the predicted proteins were different from B. subtilisFtsL. However, the ftsL gene from Staphylococcus aureus (whose product has 73% of its amino acids different from those of the B. subtilis ftsL product) was not functional. We conclude that FtsL is a highly malleable protein that can accommodate a large number of sequence changes without loss of function.

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