Global nutritional profiling for mutant and chemical mode-of-action analysis in filamentous fungi

We describe a method for gene function discovery and chemical mode-of-action analysis via nutrient utilization using a high throughput Nutritional Profiling platform suitable for filamentous microorganisms. We have optimized the growth conditions for each fungal species to produce reproducible optical density growth measurements in microtiter plates. We validated the Nutritional Profiling platform using a nitrogen source utilization assay to analyze 21 Aspergillus nidulans strains with mutations in the master nitrogen regulatory gene, areA. Analysis of these data accurately reproduced expected results and provided new data to demonstrate that this platform is suitable for fine level phenotyping of filamentous fungi. Next, we analyzed the differential responses of two fungal species to a glutamine synthetase inhibitor, illustrating chemical mode-of-action analysis. Finally, a comparative phenotypic study was performed to characterize carbon catabolite repression in four fungal species using a carbon source utilization assay. The results demonstrate differentiation between two Aspergillus species and two diverse plant pathogens and provide a wealth of new data on fungal nutrient utilization. Thus, these assays can be used for gene function and chemical mode-of-action analysis at the whole organism level as well as interspecies comparisons in a variety of filamentous fungi. Additionally, because uniform distribution of growth within wells is maintained, comparisons between yeast and filamentous forms of a single organism can be performed.Electronic Supplementary Material Supplementary material is available in the online version of this article at The revised version of the PDF file was published online in January 2004. The figures are now in color.An erratum to this article can be found at     

A new effective assay to detect antimicrobial activity of filamentous fungi

The search for new antimicrobial compounds and the optimization of production methods turn the use of antimicrobial susceptibility tests a routine. The most frequently used methods are based on agar diffusion assays or on dilution in agar or broth. For filamentous fungi, the most common antimicrobial activity detection methods comprise the co-culture of two filamentous fungal strains or the use of fungal extracts to test against single-cell microorganisms. Here we report a rapid, effective and reproducible assay to detect fungal antimicrobial activity against single-cell microorganisms. This method allows an easy way of performing a fast antimicrobial screening of actively growing fungi directly against yeast. Because it makes use of an actively growing mycelium, this bioassay also provides a way for studying the production dynamics of antimicrobial compounds by filamentous fungi. The proposed assay is less time consuming and introduces the innovation of allowing the direct detection of fungal antimicrobial properties against single cell microorganisms without the prior isolation of the active substance(s). This is particularly useful when performing large screenings for fungal antimicrobial activity. With this bioassay, antimicrobial activity of Hypholoma fasciculare against yeast species was observed for the first time.     

Growth mechanisms and growth kinetics of filamentous microorganisms

Filamentous microorganisms are of major biotechnological importance, being responsible for production of the majority of secondary metabolites, particularly antibiotics. Two main groups are involved, filamentous fungi and filamentous actinomycetes, particularly the streptomycetes. In terms of cellular growth mechanisms, these groups differ greatly. Eukaryotic fungi possess subcellular organelles and cytoskeletal structures directing growth while prokaryotic streptomycetes have no such cellular organization. Despite these fundamental differences, both groups exhibit similar morphologies, growth patterns, growth forms, and hyphal and mycelial growth kinetics on solid media and in liquid culture both grow as dispersed mycelia and pellets. The article therefore discusses the relationship between cellular growth mechanisms and vegetative growth in both filamentous fungi and actinomycetes, the conceptual and theoretical models applicable to both groups, and the significance of such models in industrial fermentation processes.     

Application of Microautoradiography to the Study of Substrate Uptake by Filamentous Microorganisms in Activated Sludge

Excessive growth of filamentous microorganisms in activated-sludge treatment plants is a major operational problem which causes poor settlement of activated sludge. An enhanced understanding of the factors controlling growth of different filamentous microorganisms is necessary in order to establish more successful control strategies. In the present study, the in situ substrate uptake was investigated by means of microautoradiography. It was demonstrated that the uptake of labeled organic substrates by the filamentous microorganisms, during short-term incubation, could be detected by microautoradiography. Viability and respiratory activity of the filaments were also detected by reduction of CTC (5-cyano-2,3-ditolyl tetrazolium chloride) and by incorporation of [(sup3)H]thymidine. Gram, Neisser, and fluorescence staining techniques were used for the localization and identification of the filaments. Activated-sludge samples from five wastewater treatment plants with bulking problems due to filamentous microorganisms were investigated. Microthrix parvicella, Nostocoida limicola, and Eikelboom's type 0041 and type 021N were investigated for their ability to take up organic substrates. A panel of six substrates, i.e., [(sup14)C]acetate, [(sup3)H]glucose, [(sup14)C]ethanol, [(sup3)H]glycine, [(sup3)H]leucine, and [(sup3)H]oleic acid, was tested. The uptake response was found to be very specific not only between the different filamentous types but also among filaments of the same type from different treatment plants. Interestingly, M. parvicella consistently took up only oleic acid among the tested substrates. It is concluded that microautoradiography is a useful method for investigation of in situ substrate uptake by filamentous microorganisms in activated sludge.     

A simple plate-assay for the screening of l-malic acid producing microorganisms

A simple plate-assay has been developed to screen microorganisms for L-malic acid production. Acid producing organisms were identified, after microbial colony growth on media containing glucose or fumaric acid as sole carbons sources, by formation of a dark halo of formazan. The halo was observed when the plate was covered with a soft agar overlay containing NAD(+)-malate dehydrogenase, NAD+, phenazine methosulfate (PMS) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). The assay developed is simple, specific for L-malic acid and therefore can be used to identify L-malic acid producing filamentous fungi using glucose as carbon source (e.g. Aspergillus strains). The assay is also applicable for screening bacteria with high fumarase activity, able to convert fumaric acid to L-malic acid.     

The half-saturation coefficient for dissolved oxygen: A dynamic method for its determination and its effect on dual species competition

A simple approach was developed to determine the half-saturation coefficient for dissolved oxygen (K(DO)) for three bacteria by maintaining a constant oxygen concentration in continuous culture, and employing a dynamic method to obtain the specific growth rate (mu) for each species. Measurement of mu at selected dissolved oxygen concentrations (DO) resulted in a typical Monod curve for a plot of mu vs. DO. Values for K(DO) and mu(max) were obtained from the Lineweaver-Burk reciprocal plot. The bacteria studied included representative strains of three microorganisms isolated in pure culture from poorly settling activated sludge: two filamentous microorganisms, Sphaerotilus natans and a second Sphaerotilus sp., and an unidentified floc-forming microorganism. The K(DO) values obtained for Sphaerotilus sp., S. natans, and the floc former were 0.014, 0.033, and 0.073 mg/L, respectively. Dual species competition experiments were conducted in continuous culture under low and high DO conditions. Successful growth competition by these microorganisms under DO-limiting conditions was consistent with experimentally determined K(DO) values. The finding of lower K(DO) values for the two Sphaerotilus species, compared to the floc former, confirmed the hypothesis that these filamentous microorganisms can outgrow floc-forming microorganisms in activated sludge when DO in the aeration basin is low.     

Extracellular cyanobacterial substances inhibit microbial growth.

Cyanobacteria are able to produce extracellular substances with different biological activities and behaviors. The marine cyanobacteria Anabaena sp. strain Hi 26 and Oscillatoria subtilissima strain Bo 62 cause significant color changes in their growth media, while viscosity of the medium is influenced by Rivularia sp. strain Bo 85 and Oscillatoria limnetica strain Flo 1. Sterile-filtered media, pregrown with the organisms mentioned above, were used to study the influence of changes in media bioactivity induced by "excreted substances" on the growth and/or morphological development of five related filamentous cyanobacterial species and on selected heterotrophic microorganisms. Cell lysis, empty sheaths, different lengths of filaments, or even single cells and a decrease in chlorophyll a and protein content were the characteristic changes obtained by such a "cyanobacterial assay." The use of a "precultured" medium, as demonstrated in an "agar diffusion assay," affects in varying degrees the growth of gram-positive and gram-negative heterotrophic bacteria, as well as of the yeast Saccharomyces cerevisiae.     

Agrichemical impact on growth and survival of non-target apple phyllosphere microorganisms

The impact of conventional agrichemicals commonly used in New Zealand apple production on non-target, culturable phyllosphere microbial populations was studied in the laboratory (agar, leaf, and seedling assays) and field (apple orchard). Morphologically distinct bacteria (three), yeasts (five), and filamentous microfungi (two) were used as indicator species. The agar assay showed that agrichemical toxicity to microorganisms was dependent on product type, product rate, and organism studied. While the fungicides metiram and captan stopped or severely reduced growth of nearly all microorganisms studied, the insecticides tebufenozide and lufenuron and the fungicide nitrothal-isopropyl showed the least amount of microbial toxicity, each affecting 2 of the 10 indicator organisms studied. In the leaf assay a single agrichemical application at field rate either reduced or increased microbial population counts, again depending on product and microorganism. Repeated agrichemical applications, however, reduced microbial population numbers from 10- to 10,000-fold in planta. Further field research validated these findings, although differences in microbial numbers before and after agrichemical applications were less dramatic. In the orchard, total organism numbers recovered within 2-6 days, but species richness (sum of recognizable taxonomic units) declined. Agrichemicals clearly affected non-target, culturable surface microorganisms. The importance of diversity and stability of microbial populations for disease control still needs to be established.     

Effect of antioxidants, pro-oxidants, respiration inhibitors and substrates on the formation of autonomic secondary colonies of Fusarium bulbigenum var. blasticola

Autonomous secondary colonies are formed on the primary colonies of Fusarium bulbigenum var. blasticola grown in a synthetic Rieder medium. The secondary colonies are similar to genuine neoplasia in the following characteristics: dedifferentiation of the mycelium filamentous structure to yield single yeast-like cells, selective growth, faulty differentiation. Just as antioxidants and respiration substrates inhibit tumour formation and growth in higher organisms, they either prevent or inhibit the formation of secondary colonies in microorganisms. In contrast, their formation is stimulated when prooxidants and respiration inhibitors are added to the growth medium.     

Microparticle-enhanced cultivation of filamentous microorganisms: increased chloroperoxidase formation by Caldariomyces fumago as an example

Microparticle-enhanced cultivation (MPEC) was applied as a novel method for improved biomass and product formation during cultivation of filamentous microorganisms. Exemplarily, chloroperoxidase (CPO) formation by Caldariomyces fumago was analyzed in the presence and absence of microparticles of different size. Particles of approximately 500 microm in diameter had no effect on growth morphology or productivity of CPO formation by C. fumago. In contrast particles of < or =42 microm in diameter led to the dispersion of the C. fumago mycelia up to the level of single hyphae. Under these conditions the maximum specific productivity of CPO formation was enhanced about fivefold and an accumulated CPO activity in the culture supernatant of more than 1,000 U mL(-1) was achieved after 10-12 days of cultivation. In addition, the novel cultivation method also showed a positive effect on growth characteristics of other filamentous microorganisms proven by the stimulation of single hyphae/cell formation.     

Microparticle based morphology engineering of filamentous microorganisms for industrial bio-production

Filamentous microorganisms are important work horses in industrial biotechnology and supply enzymes, antibiotics, pharmaceuticals, bulk and fine chemicals. Here we highlight recent findings on the use of microparticles in the cultivation of filamentous bacteria and fungi, with the aim of enabling a more precise control of their morphology towards better production performance. First examples reveal a broad application range of microparticle based processes, since multiple filamentous organisms are controllable in their growth characteristics and respond by enhanced product formation.     

Growth Inhibition of Various Organisms by a Violet Pigment,Nostocine A,Produced by Nostoc spongiaeforme

A freshwater filamentous cyanobacterium, Nostoc spongiaeforme TISTR 8169, produced and excreted a violet pigment, named nostocine A, in the culture medium. Nostocine A inhibited the growth of some typical strains of microorganisms, algae, cultured plants, and established animal cell lines.     

木质纤维素降解酶生产菌株的遗传改造及应用

通过纤维素酶将木质纤维素向生物新能源的转化对经济社会的可持续发展具有重要意义,被用于纤维素酶制剂工业化生产的微生物大多属于丝状真菌,但丝状真菌的遗传操作困难,且纤维素酶诱导机制尚未阐明,严重制约了纤维素酶高产菌株选育与应用。本文综述了近年来纤维素酶高产菌株遗传操作方法的进展,重点论述了丝状真菌合成纤维素酶过程中的信号感应、信号传导、转录调控的研究,通过理性改造以提高纤维素酶生产菌株的产酶能力,并且总结展望了丝状真菌在工业生产中的应用。     

替考游动放线菌发酵生产替考拉宁的动力学模型

根据丝状菌的生长机理和替考拉宁的发酵动力学特征提出了一个简化的形态学结构模型,并用于描述替考拉宁的发酵过程．模型将菌丝分为三种类型：具有生长活性的菌丝(G)、具有代谢活性的菌丝(N)和失活的菌丝(D)．菌丝G可因失去生长活性而转化为菌丝N,菌丝N可因失去代谢活性而转化为菌丝D．菌丝G与菌丝生长有关,而菌丝N则与替考拉宁的合成有关．该模型能较好地描述替考拉宁发酵过程、     

Williopsis californica, Williopsis saturnus, and Pachysolen tannophilus: novel microorganisms for stereoselective oxidation of secondary alcohols

A screening of 416 microorganisms from different taxonomical groups (bacteria, actinomycetes, yeasts, and filamentous fungi) has been performed looking for active strains in the stereoselective oxidation of secondary alcohols. The working collection was composed of 71 bacterial strains, 45 actinomycetes, 59 yeasts, 60 basidiomycetes, 33 marine fungi, and 148 filamentous fungi. All microorganisms selected were mesophilic. Yeasts were the most active microbial group in the whole-cell-catalyzed oxidation. Williopsis californica, Williopsis saturnus, and Pachysolen tannophilus were the strains of greatest interest, both as growing cells and as resting cells. The oxidation of the alcohols takes place when cells are in the stationary growth phase (after 48 h of culture). These three strains are S-stereoselective for the oxidation of racemic secondary alkanols and show stereospecificity in the oxidation of menthol or neo-menthol, whereas iso-menthol is not oxidized. In the case of the 1-tetrahydronaphtol enantiomers, only the S-enantiomer is oxidized. The three strains were immobilized by entrapment using agarose and agar from algae of the Gracilaria genus. The agarose derivatives displayed significant improvement in the stereospecificity of the reactions.     

Evaluation of the SOS/umu-test post-treatment assay for the detection of genotoxic activities of pure compounds and complex environmental mixtures

This study presents an evaluation of the SOS/umu-test after introducing an additional dilution and incubation in the post-treatment assay. This treatment reduces the influence of coloured test compounds that otherwise affect the colorimetric determination of the beta-galactosidase activity and the bacterial growth measurement during the testing of complex environmental samples. The post-treatment assay significantly increased the beta-galactosidase activity and consequently the enzyme induction ratios at higher doses of model genotoxins 4-nitroquinoline-N-oxide, N-methyl-N'-nitro-N-nitrosoguanidine, 2-aminoanthracene, benzo(a)pyrene with low or no effect on the sensitivity of the test itself. On the other hand tests of environmental extracts indicated significant increases in sensitivity after additional incubation. 4-Nitroquinoline-N-oxide treatments of bacteria in the test affected cell division and caused filamentous growth. The size of filamentous bacteria and incidence rate of the length categories was positively correlated with the concentrations of genotoxins. Presence of filamentous tester bacteria proved induction of SOS response and genotoxic activity of environment samples in SOS/umu-test.     

A mathematical model for the growth of mycelial pellet populations

In liquid culture, filamentous organisms often grow in the form of pellets. Growth result in an increase in radius, whereas shear forces result in release of hyphal fragments which act as centers for further pellet growth and development. A previously published model for pellet growth of filamentous microorganisms has been examined and is found to be unstable for certain parameter values. This instability has been identified as being due to inaccuracies in estimating the numbers of fragments which seed the pellet population. A revised model has been formulated, based on similar premises, but adopting a finite element approach. This considers the population of pellets to be distributed in a range of size classes. Growth results in movement to classes of increasing pellet size, while fragments enter the smallest size class, from which they grow to form further pellets. The revised model is stable and predicts changes in the distribution of pellet sizes within a population growing in liquid batch culture. It considers pellet growth and death, with fragmentation providing new centers of growth within the pellet population, and predicts the effects of shear forces on pellet growth and size distribution. Predictions of pellet size distributions are tested using previously published data on the growth of fungal pellets and further predictions are generated which are suitable for experimental testing using cultures of filamentous fungi or actinomycetes. (c) 1995 John Wiley & Sons, Inc.     

Microbial populations associated with the surface of the brown algaAscophyllum nodosum

The microorganisms on the surface of the brown algaAscophyllum nodosum, collected from an intertidal area in Nahant, Massachusetts, were examined using scanning electron microscopy. Differences in the microbial populations on the holdfast, internodal regions of the stipe, and the apical tips were apparent. The populations ranged from a lawn of end-attached bacteria above the holdfast to microcolonies of yeast cells near the apical tips. The greatest diversity of microorganisms was noted in the internodal region representing the fourth year of growth where a dense lawn of end-attached bacteria was overlaid by filamentous bacteria, pennate diatoms, and filamentous blue-green algae. A simple procedure was developed to estimate the number of bacteria on the surface of the seaweed using the scanning electron microscope. The observed distribution of epiphytes may be explained in terms of the age of the algal surface, differences in light intensity, and the differential secretion of tannin by various parts ofAscophyllum.