Key amino acid residues of the AGT1 permease required for maltotriose consumption and fermentation by Saccharomyces cerevisiae.

AIMS: The AGT1 gene encodes for a general α-glucoside-H+ symporter required for efficient maltotriose fermentation by Saccharomyces cerevisiae. In the present study, we analysed the involvement of four charged amino acid residues present in this transporter that are required for maltotriose consumption and fermentation by yeast cells. METHODS AND RESULTS: By using a knowledge-driven approach based on charge, conservation, location, three-dimensional (3D) structural modelling and molecular docking analysis, we identified four amino acid residues (Glu-120, Asp-123, Glu-167 and Arg-504) in the AGT1 permease that could mediate substrate binding and translocation. Mutant permeases were generated by site-directed mutagenesis of these charged residues, and expressed in a yeast strain lacking this permease (agt1∆). While mutating the Arg-504 or Glu-120 residues into alanine totally abolished (R504A mutant) or greatly reduced (E120A mutant) maltotriose consumption by yeast cells, as well as impaired the active transport of several other α-glucosides, in the case of the Asp-123 and Glu-167 amino acids, it was necessary to mutate both residues (D123G/E167A mutant) in order to impair maltotriose consumption and fermentation. CONCLUSIONS: Based on the results obtained with mutant proteins, molecular docking and the localization of amino acid residues, we propose a transport mechanism for the AGT1 permease. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results present new insights into the structural basis for active α-glucoside-H+ symport activity by yeast transporters, providing the molecular bases for improving the catalytic properties of this type of sugar transporters.

Extracellular maltotriose hydrolysis by Saccharomyces cerevisiae cells lacking the AGT1 permease.

In brewing, maltotriose is the least preferred sugar for uptake by Saccharomyces cerevisiae cells. Although the AGT1 permease is required for efficient maltotriose fermentation, we have described a new phenotype in some agt1Δ strains of which the cells do not grow on maltotriose during the first 3-4 days of incubation, but after that, they start to grow on the sugar aerobically. Aiming to characterize this new phenotype, we performed microarray gene expression analysis which indicated upregulation of high-affinity glucose transporters (HXT4, HXT6 and HXT7) and α-glucosidases (MAL12 and IMA5) during this delayed cellular growth. Since these results suggested that this phenotype might be due to extracellular hydrolysis of maltotriose, we attempted to detect glucose in the media during growth. When an hxt-null agt1Δ strain was grown on maltotriose, it also showed the delayed growth on this carbon source, and glucose accumulated in the medium during maltotriose consumption. Considering that the poorly characterized α-glucosidase encoded by IMA5 was among the overexpressed genes, we deleted this gene from an agt1Δ strain that showed delayed growth on maltotriose. The ima5Δ agt1Δ strain showed no maltotriose utilization even after 200 h of incubation, suggesting that IMA5 is likely responsible for the extracellular maltotriose hydrolysis. SIGNIFICANCE AND IMPACT OF THE STUDY: Maltotriose is the second most abundant sugar present in brewing. However, many yeast strains have difficulties to consume maltotriose, mainly because of its low uptake rate by the yeast cells when compared to glucose and maltose uptake. The AGT1 permease is required for efficient maltotriose fermentation, but some strains deleted in this gene are still able to grow on maltotriose after an extensive lag phase. This manuscript shows that such delayed growth on maltotriose is a consequence of extracellular hydrolysis of the sugar. Our results also indicate that the IMA5-encoded α-glucosidase is likely the enzyme responsible for this phenotype.

Secretion of the acid trehalase encoded by the CgATH1 gene allows trehalose fermentation by Candida glabrata.

The emergent pathogen Candida glabrata differs from other yeasts because it assimilates only two sugars, glucose and the disaccharide trehalose. Since rapid identification tests are based on the ability of this yeast to rapidly hydrolyze trehalose, in this work a biochemical and molecular characterization of trehalose catabolism by this yeast was performed. Our results show that C. glabrata consumes and ferments trehalose, with parameters similar to those observed during glucose fermentation. The presence of glucose in the medium during exponential growth on trehalose revealed extracellular hydrolysis of the sugar by a cell surface acid trehalase with a pH optimum of 4.4. Approximately ∼30% of the total enzymatic activity is secreted into the medium during growth on trehalose or glycerol. The secreted enzyme shows an apparent molecular mass of 275 kDa in its native form, but denaturant gel electrophoresis revealed a protein with ∼130 kDa, which due to its migration pattern and strong binding to concanavalin A, indicates that it is probably a dimeric glycoprotein. The secreted acid trehalase shows high affinity and activity for trehalose, with Km and Vmax values of 3.4 mM and 80 U (mg protein)(-1), respectively. Cloning of the CgATH1 gene (CAGLOK05137g) from de C. glabrata genome, a gene showing high homology to fungal acid trehalases, allowed trehalose fermentation after heterologous expression in Saccharomyces cerevisiae.

Characterization of maltotriose transporters from the Saccharomyces eubayanus subgenome of the hybrid Saccharomyces pastorianus lager brewing yeast strain Weihenstephan 34/70.

The genome from the Saccharomyces pastorianus industrial lager brewing strain Weihenstephan 34/70, a natural Saccharomyces cerevisiae/Saccharomyces eubayanus hybrid, indicated the presence of two different maltotriose transporter genes: a new gene in the S. eubayanus subgenome with 81% of homology to the AGT1 permease from S. cerevisiae, and an amplification of the S. eubayanus MTY1 maltotriose permease previously identified in S. pastorianus yeasts. To characterize these S. eubayanus transporter genes, we used a S. cerevisiae strain deleted in the AGT1 permease and introduced the desired permease gene(s) into this locus through homologous recombination. Our results indicate that both the MTY1 and AGT1 genes from the S. eubayanus subgenome encode functional maltotriose transporters that allow fermentation of this sugar by yeast cells, despite their apparent differences in the kinetics of maltotriose-H(+) symport activity. The presence of two maltotriose transporters in the S. eubayanus subgenome not only highlights the importance of sugar transport for efficient maltotriose utilization by industrial yeasts, but these new genes can be used in breeding and/or selection programs aimed at increasing yeast fitness for the efficient fermentation of brewer's wort.

Outbreak of Neisseria meningitidis C in workers at a large food-processing plant in Brazil: challenges of controlling disease spread to the larger community.

SUMMARYAn outbreak of meningococcal disease (MD) with severe morbidity and mortality was investigated in midwestern Brazil in order to identify control measures. A MD case was defined as isolation of Neisseria meningitidis, or detection of polysaccharide antigen in a sterile site, or presence of clinical purpura fulminans, or an epidemiological link with a laboratory-confirmed case-patient, between June and August 2008. In 8 out of 16 MD cases studied, serogroup C ST103 complex was identified. Five (31%) cases had neurological findings and five (31%) died. The attack rate was 12 cases/100 000 town residents and 60 cases/100 000 employees in a large local food-processing plant. We conducted a matched case-control study of eight primary laboratory-confirmed cases (1:4). Factors associated with illness in single variable analysis were work at the processing plant [matched odds ratio (mOR) 22, 95% confidence interval (CI) 2·3-207·7, P<0·01], and residing <1 year in Rio Verde (mOR 7, 95% CI 1·11-43·9, P<0·02). Mass vaccination (>10 000 plant employees) stopped propagation in the plant, but not in the larger community.

Microarray karyotyping of maltose-fermenting Saccharomyces yeasts with differing maltotriose utilization profiles reveals copy number variation in genes involved in maltose and maltotriose utilization.

AIMS: We performed an analysis of maltotriose utilization by 52 Saccharomyces yeast strains able to ferment maltose efficiently and correlated the observed phenotypes with differences in the copy number of genes possibly involved in maltotriose utilization by yeast cells. METHODS AND RESULTS: The analysis of maltose and maltotriose utilization by laboratory and industrial strains of the species Saccharomyces cerevisiae and Saccharomyces pastorianus (a natural S. cerevisiae/Saccharomyces bayanus hybrid) was carried out using microscale liquid cultivation, as well as in aerobic batch cultures. All strains utilize maltose efficiently as a carbon source, but three different phenotypes were observed for maltotriose utilization: efficient growth, slow/delayed growth and no growth. Through microarray karyotyping and pulsed-field gel electrophoresis blots, we analysed the copy number and localization of several maltose-related genes in selected S. cerevisiae strains. While most strains lacked the MPH2 and MPH3 transporter genes, almost all strains analysed had the AGT1 gene and increased copy number of MALx1 permeases. CONCLUSIONS: Our results showed that S. pastorianus yeast strains utilized maltotriose more efficiently than S. cerevisiae strains and highlighted the importance of the AGT1 gene for efficient maltotriose utilization by S. cerevisiae yeasts. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results revealed new maltotriose utilization phenotypes, contributing to a better understanding of the metabolism of this carbon source for improved fermentation by Saccharomyces yeasts.

Improvement of maltotriose fermentation by Saccharomyces cerevisiae.

AIMS: To enhance the fermentation of maltotriose by industrial Saccharomyces cerevisiae strains. METHODS AND RESULTS: The capability to ferment maltotriose by an industrial yeast strain that uses this sugar aerobically was tested in shake flasks containing rich medium. While the presence of maltose in the medium did not improve maltotriose fermentation, enhanced and constitutive expression of the AGT1 permease not only increased the uptake of maltotriose, but allowed efficient maltotriose fermentation by this strain. Supplementation of the growth medium with 20 mmol magnesium l(-1) also increased maltotriose fermentation. CONCLUSIONS: Over expression of the AGT1 permease and magnesium supplementation improved maltotriose fermentation by an industrial yeast strain that respired but did not ferment this sugar. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes to the elucidation of the roles of the AGT1 permease and nutrients in the fermentation of all sugars present in starch hydrolysates, a highly desirable trait for several industrial yeasts.

The expanding role of the clinical coordinator.

This article describes the role of the clinical coordinator in nurse anesthesia educational programs, a role which has greatly expanded as programs shift to university-based frameworks with multiple clinical sites. The role of the clinical coordinator has become vital in the success of schools of nurse anesthesia based in the university/college setting. The significance and functional responsibilities of this role need to be broadened by making the clinical coordinator a university appointed representative. The expanded role provides for university representation at each site, improves the lines of communication between the university and each site, and allows for regular visits to each clinical site. Hopefully, program directors, faculty members, and students will benefit from this innovative attempt at bridging the gap between the university and the associated clinical facilities. Regional coordinators also play a vital role in coordinating student functions between various institutions and the clinical coordinators in each of these institutions, as well as representing the university/school of nurse anesthesia interest and needs in these settings.

Cardiovascular changes associated with aging: the anesthetic implications.

Successful anesthesia care of the elderly patient is highly dependent on the anesthesia care provider's knowledge of the physiological alterations on the cardiovascular system associated with aging. It is projected that by the year 2000, the elderly segment of the population will increase to 13% and by the year 2030, 52 million Americans, or 17% of the population will be older than age 65. More people than ever before are reaching old age, with the latest statistics indicating that life expectancy for a man of 45 years has increased from 70.4 to 77.3 years and from 77.0 to 82.8 years for a woman. One of the primary factors associated with the increase in longevity is the decrease in mortality from cardiovascular diseases. Since the 1960s, there has been a decline in cardiovascular mortality of about 3% per year. Recent technological advances and healthier lifestyles are among the reasons contributing to this trend. With these positive changes impacting the elderly population, anesthesia care providers must become better prepared to select and administer the appropriate agents that will ultimately influence perioperative outcome.

Miocardite primaria aguda.Estudo a longo prazo de sete criancas. / Acute primary myocarditis. Long-term study of seven children

Os autores fizeram uma revisao da miocardite primaria na crianca e destacam que desde 1973 tiveram a oportunidade de observar 19 criancas portadoras desta patologia, sendo que somente sete foram incluidas nesta revisao, pois as demais tiveram o inicio da doenca, ou seja, sua fase aguda, em epoca anterior ao inicio deste trabalho. Comentam, tambem, os aspectos clinicos, radiologicos, eletrocardiograficos e terapeuticos e discutem as diferentes opinioes quanto ao uso, ou nao, da corticoterapia; dao enfase especial a evolucao e relatam que duas criancas evoluiram para a cura, quatro para uma forma cronica e uma para o obito. Todos os pacientes com evolucao cronica foram observados durante quatro anos e, progressivamente, no ECG, passaram a evidenciar sinais de acentuada hipertrofia ventricular esquerda com ondas Q importantes em V5 e V6. Frente a estes achados os autores comecaram os recentes trabalhos que indicam a possibilidade da fibroelastose endocardica ser determinada por uma previa miocardite a virus