Marine killer yeasts active against a yeast strain pathogenic to crab Portunus trituberculatus

Some marine yeasts have recently been recognised as pathogenic agents in crab mariculture, but may be inhibited or killed by 'killer' yeast strains. We screened multiple yeast strains from seawater, sediments, mud of salterns, guts of marine fish, and marine algae for killer activity against the yeast Metchnikowia bicuspidata WCY (pathogenic to crab Portunus trituberculatus), and found 17 strains which could secrete toxin onto the medium and kill the pathogenic yeast. Of these, 5 strains had significantly higher killing activity than the others; routine identification and molecular methods showed that these were Williopsis saturnus WC91-2, Pichia guilliermondii GZ1, Pichia anomala YF07b, Debaryomyces hansenii hcx-1 and Aureobasidium pullulans HN2.3. We found that the optimal conditions for killer toxin production and action of killer toxin produced by the marine killer yeasts were not all in agreement with those of marine environments and for crab cultivation. We found that the killer toxins produced by the killer yeast strains could kill other yeasts in addition to the pathogenic yeast, and NaCl concentration in the medium could change killing activity spectra. All the crude killer toxins produced could hydrolyze laminarin and the hydrolysis end products were monosaccharides.     

Yeast killer toxins,molecular mechanisms of their action and their applications

Killer toxins secreted by some yeast strains are the proteins that kill sensitive cells of the same or related yeast genera. In recent years, many new yeast species have been found to be able to produce killer toxins against the pathogenic yeasts, especially Candida albicans. Some of the killer toxins have been purified and characterized, and the genes encoding the killer toxins have been cloned and characterized. Many new targets including different components of cell wall, plasma membrane, tRNA, DNA and others in the sensitive cells for the killer toxin action have been identified so that the new molecular mechanisms of action have been elucidated. However, it is still unknown how some of the newly discovered killer toxins kill the sensitive cells. Studies on the killer phenomenon in yeasts have provided valuable insights into a number of fundamental aspects of eukaryotic cell biology and interactions of different eukaryotic cells. Elucidation of the molecular mechanisms of their action will be helpful to develop the strategies to fight more and more harmful yeasts.     

Conversion of pentoses by yeasts

The utilization and conversion of D-xylose, D-xylulose, L-arabinose, and xylitol by yeast strains have been investigated with the following results: (1) The majority of yeasts tested utilize D-xylose and produce polyols, ethanol, and organic acids. The type and amount of products formed varies with the yeast strains used. The most commonly detected product is xylitol. (2)The majority of yeasts tested utilize D-xylulose aerobically and fermentatively to produce ethanol, xylitol, D-arabitol, and organic acids. The type and amount of products varies depending upon the yeast strains used. (3) Xylitol is a poor carbon and energy source for most yeasts tested. Some yeast strains produce small amounts of ethanol from xylitol. (4) Most yeast strains utilize L-arabinose, and L-arabitol is the common product. Small amounts of ethanol are also produced by some yeast strains. (5) Of the four substrates examined, D-xylulose was the perferred substrate, followed by D-xylose, L-arabinose, and xylitol. (6) Mutant yeast strains that exhibit different metabolic product patterns can be induced and isolated from Candida sp. Saccharomyces cerevisiae, and other yeasts. These mutant strains can be used for ethanol production from D-xylose as well as for the study of metabolic regulation of pentose utilization in yeasts.     

Production,characterization and gene cloning of the extracellular enzymes from the marine-derived yeasts and their potential applications

In this review article, the extracellular enzymes production, their properties and cloning of the genes encoding the enzymes from marine yeasts are overviewed. Several yeast strains which could produce different kinds of extracellular enzymes were selected from the culture collection of marine yeasts available in this laboratory. The strains selected belong to different genera such as Yarrowia, Aureobasidium, Pichia, Metschnikowia and Cryptococcus. The extracellular enzymes include cellulase, alkaline protease, aspartic protease, amylase, inulinase, lipase and phytase, as well as killer toxin. The conditions and media for the enzyme production by the marine yeasts have been optimized and the enzymes have been purified and characterized. Some genes encoding the extracellular enzymes from the marine yeast strains have been cloned, sequenced and expressed. It was found that some properties of the enzymes from the marine yeasts are unique compared to those of the homologous enzymes from terrestrial yeasts and the genes encoding the enzymes in marine yeasts are different from those in terrestrial yeasts. Therefore, it is of very importance to further study the enzymes and their genes from the marine yeasts. This is the first review on the extracellular enzymes and their genes from the marine yeasts.     

Experimentally induced marine flexibacteriosis in Atlantic salmon smolts Salmo salar. I. Pathogenicity

Tenacibaculum maritimum causes marine flexibacteriosis in many cultured fish species, including Atlantic salmon Salmo salar in Tasmania, Australia. Several aspects of the pathogenicity of this bacterium were investigated in naive Atlantic salmon smolts using different isolates, growth conditions and doses to produce a model of infection. We found that T. maritimum is pathogenic to Atlantic salmon using either marine Shieh's or marine Ordal's culture medium. The use of aeration in broth culture produced a dose effect in challenge due to a 'clumping' of the bacteria during culture. The virulence of a strain appears to be connected with this 'clumping'; the more adherent the cells, the more pathogenic the strain. Differences in virulence between 3 strains was apparent, with 1 of the strains (89/4747) being non-pathogenic and unable to produce disease in the host. The 2 other strains (89/4762, 00/3280) were highly virulent, resulting in 100% mortalities within 3 d. A reproducible model of infection has been established in the present study using strain 89/4762. Results from the present study provide a better insight into the nature of the disease.     

Mannose-containing polysaccharides of yeasts

The known species of yeasts produce at lest 180 different polysaccharides having mannose as a major constituent. The type of mannan formed is characteristic of the yeast species, and can be used as an aid in identification and classification. The immunological characteristics of yeasts depend largely upon the nature of the mannans. Some of the yeast mannans can be produced in relatively high yields, and have been suggested as replacements for plant and bacterial gums as thickeners, dispersing agents, and similar applications. However, more significant uses, based on the finer differences in their chemical structures and the specific reactions they undergo, may result from a closer study of these polymers.     

Occurrence and Diversity of Yeasts in the Mid-Atlantic Ridge Hydrothermal Fields Near the Azores Archipelago

The yeast community associated with deep-sea hydrothermal systems of the Mid-Atlantic Rift was surveyed for the first time. This study relied on a culture-based approach using two different growth media: a conventional culture medium for yeasts supplemented with sea salts (MYPss) and the same medium additionally supplemented with sulfur (MYPssS). For the evaluation of species diversity, a molecular approach involving minisatellite-primed polymerase chain reaction (MSP-PCR) strain typing and sequence analysis of the D1/D2 domains of the 26S rDNA was followed. In the seven water samples that were studied, the number of colony-forming units per liter (cfu/L) ranged from 0 to 5940. The nonpigmented yeasts were much more abundant than the pink-pigmented ones. This disproportion was not observed in studies of other marine systems and may be due to the unique conditions of hydrothermal vents, characterized by a rich animal and microbial diversity and therefore by the availability of organic compounds utilizable by yeasts. Higher counts of nonpigmented yeast were obtained using MYPss, whereas for pink yeasts, higher counts were obtained using MYPssS. Moreover, among pink yeasts, some of the MSP-PCR classes obtained were composed of isolates obtained only on MYPssS, which might be an indication that these isolates are adapted to the ecosystems of the hydrothermal vents. Twelve phylotypes belonged to the Ascomycota and seven phylotypes belonged to the Basidiomycota. The nonpigmented yeasts were identified as Candida atlantica, C. atmosphaerica, C. lodderae, C. parapsilosis, Exophiala dermatitidis, Pichia guilliermondii, and Trichosporon dermatis, whereas the pigmented yeasts were identified as Rhodosporidium diobovatum, R. sphaerocarpum, R. toruloides, and Rhodotorula mucilaginosa. Some of the yeasts that were found belong to phylogenetic groups that include species reported from other marine environments, and eight phylotypes represent undescribed species. The new phylotypes found at Mid-Atlantic Ridge hydrothermal fields represent 33% of the total number of yeast taxa that were found.     

Disease in marine aquaculture

It has become almost a truism that success in intensive production of animals must be based in part on development of methods for disease diagnosis and control. Excellent progress has been made in methods of diagnosis for major pathogens of cultivated fish, crustacean and molluscan species. In many instances these have proved to be facultative pathogens, able to exert severe effects in populations of animals under other stresses (marginal physical or chemical conditions; overcrowding). The concept of stress management as a critical prophylactic measure is not new, but its significance is being demonstrated repeatedly. The particular relationship of water quality and facultative pathogens such asVibrio, Pseudomonas andAeromonas species has been especially apparent. Virus diseases of marine vertebrates and invertebrates — little known two decades ago — are now recognized to be of significance to aquaculture. Virus infections of oysters, clams, shrimps and crabs have been described, and mortalities have been attributed to them. Several virus diseases of fish have also been recognized as potential or actual problems in culture. In some instances, the pathogens seem to be latent in natural populations, and may be provoked into patency by stresses of artificial environments. One of the most promising approaches to disease prophylaxis is through immunization. Fish respond well to various vaccination procedures, and new non-stressing methods have been developed. Vibriosis — probably the most severe disease of ocean-reared salmon — has been controlled to a great extent through use of a polyvalent bacterin, which can be modified as new pathogenic strains are isolated. Prophylactic immunization for other bacterial diseases of cultivated fish has been attempted, especially in Japan, with some success. There is also some evidence that the larger crustaceans may be immunologically responsive, and that at least short-term protection may be afforded to cultured populations. Some progress has been made in marine disease control through chemical treatment in intensive culture systems, principally through application and modification of methods developed for freshwater aquaculture. Major constraints to use of chemicals are restrictions due to public health concerns about food contamination, and the negative effects of some chemicals on biological filters and on algal food production. There is a continuing need, however, for development of specific treatments for acute disease episodes — such as the nitrofurans, developed in Japan, which are effective for some bacterial diseases. The history of aquaculture — freshwater as well as marine — has been characterized by transfers and introductions of species to waters beyond their present ranges. The process continues, and carries with it the possibility of transfers of pathogens to native species and to the recipient culture environments. International groups are attempting to define codes of practice to govern such mass movements, but examples of introductions of real or potential pathogens already exist. The most recent and the most dramatic is the world wide transfer of a virus pathogen of penaeid shrimps. Earlier examples include the introduction of a protozoan pathogen of salmonids to the western hemisphere, and the introduction of a parasitic copepod from the Far East to the west coast of North America and to France. The conclusion is inevitable — diseases are substantial deterrents to aquaculture production. Diagnostic and control procedures are and will be important components of emerging aquaculture technology.     

Siderophore production by the marine-derived Aureobasidium pullulans and its antimicrobial activity

Over 300 yeast strains isolated from different marine environments were screened for their ability to produce siderophore. Among them, only the yeast strain HN6.2 which was identified to be Aureobasidium pullulans was found to produce high level of the siderophore. Under the optimal conditions, this yeast strain could produce 1.1 mg/ml of the siderophore. The crude siderophore produced by the yeast strain HN6.2 was able to inhibit cell growth of Vibrio anguillarum and Vibrio parahaemolyticus, isolated from the diseased marine animals.     

Oxylipin studies expose aspirin as antifungal

The presence of aspirin-sensitive 3-hydroxy fatty acids (i.e. 3-OH oxylipins) in yeasts was first reported in the early 1990s. Since then, these oxidized fatty acids have been found to be widely distributed in yeasts. 3-OH oxylipins may: (1) have potent biological activity in mammalian cells; (2) act as antifungals; and (3) assist during forced spore release from enclosed sexual cells (asci). A link between 3-OH oxylipin production, mitochondria and aspirin sensitivity exists. Research suggests that: (1) 3-OH oxylipins in some yeasts are probably also produced by mitochondria through incomplete beta-oxidation; (2) aspirin inhibits mitochondrial beta-oxidation and 3-OH oxylipin production; (3) yeast sexual stages, which are probably more dependent on mitochondrial activity, are also characterized by higher 3-OH oxylipin levels as compared to asexual stages; (4) yeast sexual developmental stages as well as cell adherence/flocculation are more sensitive to aspirin than corresponding asexual growth stages; and (5) mitochondrion-dependent asexual yeast cells with a strict aerobic metabolism are more sensitive to aspirin than those that can also produce energy through an alternative anaerobic glycolytic fermentative pathway in which mitochondria are not involved. This review interprets a wide network of studies that reveal aspirin to be a novel antifungal.     

Occurrence of human-associated yeasts in bivalve shellfish from Long Island Sound.

Candida parapsilosis, C. tropicalis, and Torulopsis glabrata were the human-associated yeasts most frequently isolated from quahogs, oysters, and mussels collected from four estuarine areas along the northern shore of Long Island Sound. Some inconsistency and seasonal variation in the occurrence of these and other yeast species were noted. In particular, C. albicans densities were greatest during colder months in the more heavily polluted waters. A total of 347 yeasts were isolated and cultured at 37 degrees C and, of these, 219 of 62% were human-associated forms. Generally, these yeasts in the animals sampled reflected the overall pollution status of the estuary from which they were taken. This study represents a clear demonstration of potentially pathogenic yeasts in a valuable marine resource.     

Occurrence of human-associated yeasts in bivalve shellfish from Long Island Sound.

Candida parapsilosis, C. tropicalis, and Torulopsis glabrata were the human-associated yeasts most frequently isolated from quahogs, oysters, and mussels collected from four estuarine areas along the northern shore of Long Island Sound. Some inconsistency and seasonal variation in the occurrence of these and other yeast species were noted. In particular, C. albicans densities were greatest during colder months in the more heavily polluted waters. A total of 347 yeasts were isolated and cultured at 37 degrees C and, of these, 219 of 62% were human-associated forms. Generally, these yeasts in the animals sampled reflected the overall pollution status of the estuary from which they were taken. This study represents a clear demonstration of potentially pathogenic yeasts in a valuable marine resource.     

The production and detoxification of a potent cytotoxin, nitric oxide, by pathogenic enteric bacteria

The nitrogen cycle is based on several redox reactions that are mainly accomplished by prokaryotic organisms, some archaea and a few eukaryotes, which use these reactions for assimilatory, dissimilatory or respiratory purposes. One group is the Enterobacteriaceae family of Gammaproteobacteria, which have their natural habitats in soil, marine environments or the intestines of humans and other warm-blooded animals. Some of the genera are pathogenic and usually associated with intestinal infections. Our body possesses several physical and chemical defence mechanisms to prevent pathogenic enteric bacteria from invading the gastrointestinal tract. One response of the innate immune system is to activate macrophages, which produce the potent cytotoxin nitric oxide (NO). However, some pathogens have evolved the ability to detoxify NO to less toxic compounds, such as the neuropharmacological agent and greenhouse gas nitrous oxide (N?O), which enables them to overcome the host's attack. The same mechanisms may be used by bacteria producing NO endogenously as a by-product of anaerobic nitrate respiration. In the present review, we provide a brief introduction into the NO detoxification mechanisms of two members of the Enterobacteriaceae family: Escherichia coli and Salmonella enterica serovar Typhimurium. These are discussed as comparative non-pathogenic and pathogenic model systems in order to investigate the importance of detoxifying NO and producing N?O for the pathogenicity of enteric bacteria.     

The costs and benefits of killer toxin production by the yeast Pichia kluyveri

Numerous yeast species in many genera are able to produce and excrete extracellular toxic proteins (mycocins) that can kill other specific sensitive yeasts. Natural distributions of killer yeasts suggest that they may be important in maintaining community composition and provide a benefit to the toxin producing cells. The fact that not all yeasts are killers and that polymorphisms exist within some killer species suggests there may be a cost associated with killer toxin production. This study focuses on the costs and benefits associated with toxin production by the yeast Pichia kluyveri. Strains differing in their ability to kill were obtained by tetrad dissection. One parent strain produced spores that exhibited a trade-off between killing ability and intrinsic growth rate. A killer clone from this strain was able to maintain a higher proportion of cells than a non-killer when grown with the same sensitive yeast under laboratory-simulated natural conditions. On the other hand, when grown with a yeast not sensitive to Pichia kluyveri toxin, the non-killer maintained a higher proportion of the total community than did the killer clone. The data support the hypothesis that there are both costs and benefits to producing killer toxin, and based on this, selection may favor different phenotypes in different conditions.     

Bioactive substances produced by marine isolates of Pseudomonas

Pseudomonas is a genus of non-fermentative gram-negative Gammaproteobacteria found both on land and in the water. Many terrestrial isolates of this genus have been studied extensively. While many produce bioactive substances, enzymes, and biosurfactants, other Pseudomonas isolates are used for biological control of plant diseases and bioremediation. In contrast, only a few marine isolates of this genus have been described that produce novel bioactive substances. The chemical structures of the bioactive substances from marine Pseudomonas are diverse, including pyroles, pseudopeptide pyrrolidinedione, phloroglucinol, phenazine, benzaldehyde, quinoline, quinolone, phenanthren, phthalate, andrimid, moiramides, zafrin and bushrin. Some of these bioactive compounds are antimicrobial agents, and dibutyl phthalate and di-(2-ethylhexyl) phthalate have been reported to be cathepsin B inhibitors. In addition to being heterogeneous in terms of their structures, the antibacterial substances produced by Pseudomonas also have diverse mechanisms of action: some affect the bacterial cell membrane, causing bacterial cell lysis, whereas others act as acetyl-CoA carboxylase and nitrous oxide synthesis inhibitors. Marine Pseudomonas spp. have been isolated from a wide range of marine environments and are a potential untapped source for medically relevant bioactive substances.     

Role of yeasts in the qualitative structuring of extra virgin olive oil

This review sought to describe the role played by some components of the microbiota of extra virgin olive oil (EVOO), particularly yeasts, in structuring the physicochemical and sensorial quality of freshly produced olive oil. Yeasts can survive during the entire storage period of the product. To date, approximately 25 yeast species isolated from oil produced in more than six countries have been identified, eight of which are classified as new species. Some yeast species improve the health qualities of oil, whereas many others improve the chemical composition and sensory characteristics based on β-glucosidase and esterase enzymes, which are involved in the hydrolysis of the bitter glucoside known as oleuropein. However, some species, which are typically favoured by the high water content in the oily matrix, such as lipase-producing yeasts, can worsen the initial chemical characteristics of EVOO oil during storage. Some physical treatments that are compatible with the EVOO production specification affect the biotic component of the oil by reducing the concentration of yeasts. The possibility of minimizing the invasive action on the biotic component of the oil by appropriately selecting the physical treatment for each oil is discussed.     

Osmolarity is an independent trigger of Acanthamoeba castellanii differentiation

Like many yeasts, bacteria, and other sporulating microorganisms, Acanthamoeba castellanii (Neff), a free-living amoeba with pathogenic relatives, differentiates into a dormant form when deprived of nutrients. Acanthamoeba cysts redifferentiate into trophozoites when food is resupplied. We report here that Acanthamoeba encystment is also triggered by elevated osmolarity, and that osmolarity and cell surface receptor binding are synergistic in triggering differentiation. Additions of sodium chloride or glucose to rich growth media were used to produce specific osmolarity increases and similar encystment results were obtained with either additive. Although many organisms, including Acanthamoeba and mammalian cells, have been shown to adapt to hyperosmolar conditions, this is the first demonstration that hyperosmolarity can be a primary differentiation signal. © 1996 Wiley-Liss, Inc.     

Yeasts in sustainable bioethanol production: A review

Bioethanol has been identified as the mostly used biofuel worldwide since it significantly contributes to the reduction of crude oil consumption and environmental pollution. It can be produced from various types of feedstocks such as sucrose, starch, lignocellulosic and algal biomass through fermentation process by microorganisms. Compared to other types of microoganisms, yeasts especially Saccharomyces cerevisiae is the common microbes employed in ethanol production due to its high ethanol productivity, high ethanol tolerance and ability of fermenting wide range of sugars. However, there are some challenges in yeast fermentation which inhibit ethanol production such as high temperature, high ethanol concentration and the ability to ferment pentose sugars. Various types of yeast strains have been used in fermentation for ethanol production including hybrid, recombinant and wild-type yeasts. Yeasts can directly ferment simple sugars into ethanol while other type of feedstocks must be converted to fermentable sugars before it can be fermented to ethanol. The common processes involves in ethanol production are pretreatment, hydrolysis and fermentation. Production of bioethanol during fermentation depends on several factors such as temperature, sugar concentration, pH, fermentation time, agitation rate, and inoculum size. The efficiency and productivity of ethanol can be enhanced by immobilizing the yeast cells. This review highlights the different types of yeast strains, fermentation process, factors affecting bioethanol production and immobilization of yeasts for better bioethanol production.     

Production of Ethanol from d-Xylose by Using d-Xylose Isomerase and Yeasts

d-Xylulose, an intermediate of d-xylose catabolism, was observed to be fermentable to ethanol and carbon dioxide in a yield of greater than 80% by yeasts (including industrial bakers' yeast) under fermentative conditions. This conversion appears to be carried out by many yeasts known for d-glucose fermentation. In some yeasts, xylitol, in addition to ethanol, was produced from d-xylulose. Fermenting yeasts are also able to produce ethanol from d-xylose when d-xylose isomerizing enzyme is present. The results indicate that ethanol could be produced from d-xylose in a yield of greater than 80% by a two-step process. First, d-xylose is converted to d-xylulose by xylose isomerase. d-Xylulose is then fermented to ethanol by yeasts.     

McRAPD as a new approach to rapid and accurate identification of pathogenic yeasts

Despite advances in antifungal prophylaxis and therapy, morbidity and mortality incurred by yeasts remain a significant burden. As pathogenic yeast species vary in their susceptibilities to antifungal agents, clinical microbiology laboratories face an important challenge to identify them rapidly and accurately. Although a vast array of phenotyping and genotyping methods has been developed, these are either unable to cover the whole spectrum of potential yeast pathogens or can do this only in a rather costly or laborious way. Random amplified polymorphic DNA (RAPD) fingerprinting was repeatedly demonstrated to be a convenient tool for species identification in pathogenic yeasts. However, its wider acceptance has been limited mainly due to special expertise and software needed for analysis and comparison of the resulting banding patterns. Based on a pilot study, we demonstrate here that a simple and rapid melting curve analysis of RAPD products can provide data for identification of five of the most medically important Candida species. We have termed this new approach melting curve of random amplified polymorphic DNA (McRAPD) to emphasize its rapidity and potential for automation, highly desirable features for a routine laboratory test.