Centre for Process Biotechnology, Department of Biotechnology, Building 223, Technical University of Denmark, 2800 Lyngby, Denmark1
Author for correspondence: Jens Nielsen. Tel: +45 4525 2694. Fax: +45 4588 4148. e-mail: jn{at}ibt.dtu.dk
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ABSTRACT |
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Keywords: morphology, Aspergillus, septation, fragmentation, metabolic activity
Abbreviations: CFW, calcofluor white; DAPI, 4,6-diamidino-2-phenylindole dihydrochloride
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INTRODUCTION |
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Elucidation of the physiological basis of the control of hyphal growth and branching is crucial if routes to the control and regulation of morphology on a molecular level are to be determined. It would be particularly desirable to identify the key genes influencing morphology in filamentous fungi as this would open the possibilities for mutant strains to be constructed with more desirable morphologies for submerged bioprocesses. Although attainment of such goals lies in the future, it is possible to facilitate this process through characterization of morphological mutants to determine the relationship between morphology, growth and behaviour of such strains in submerged cultivation.
A class of Aspergillus nidulans mutants defective in septation has been well described in the literature (Harris et al., 1994 , 1997
; Momany et al., 1995
; Harris & Hamer, 1995
) and thus provides the strains required for characterization of defined morphological mutants in submerged cultivation. A sepA mutant (sepA4
Bm) is of particular interest; it is a null mutant that does not produce septa in the first hours of growth while displaying aberrant branching patterns and abnormally wide hyphae (Harris et al., 1997
). Further, the positioning of nuclei in sepA deletion mutants is irregular; this is likely to be a result of lost control of hyphal compartment size due to the absence of septa.
Septation is an important event in the life cycle of filamentous fungi, compartmentalizing hyphal filaments as they are laid down, producing multi-nucleate cells. Septum formation requires the integration of mitotic, cytokinetic and cell-wall biosynthetic events (Momany et al., 1995 ), enabling partitioning of the filaments in a regulated manner (following subsequent rounds of mitosis). Septation also allows differentiation of fungal surface colonies. Characteristic differentiation of cellular activities is exhibited along a hyphal filament depending on the location within a colony (Smith & Anderson, 1973
) and demonstrates the heterogeneous nature of the hyphal filament.
In A. nidulans, cytokinesis (septation) is initiated through the formation of a transient actin ring at the division site; a process that is closely co-ordinated with mitosis (Harris et al., 1997 ), ensuring chromosomes and organelles are properly partitioned in the resulting compartments (Momany et al., 1995
). Septation requires nuclear division (Wolkow et al., 1996
) and in A. nidulans septation is delayed until three rounds of nuclear division have concluded (Harris & Kraus, 1998
). Nuclear division occurs only in the apical compartment (Müller et al., 2000
; King & Alexander, 1969
) with the onset of mitosis being regulated through a size mechanism relating the ratio of cytoplasmic volume to DNA (Trinci, 1979
). In A. nidulans, size regulation is likely to be post-mitotic and the critical parameter controlling the onset of cytokinesis (Wolkow et al., 1996
). The size (length) of the apical compartment increases with increasing specific growth rate (Müller et al., 2000
). The subapical compartments of a mycelium, however, have a uniform size delimited by septa, and a regulated (average) number of nuclei (Müller et al., 2000
; Wolkow et al., 1996
).
Whilst previous studies of mutants defective in septation (Harris et al., 1994 , 1997
; Harris & Hamer, 1995
; Momany et al., 1995
) or nuclear positioning (Wolkow et al., 1996
; Xiang et al., 1994
) have yielded valuable information on how the processes of cell growth, septation and mitosis are co-ordinated and regulated, the relevance of these phenotypes during submerged batch cultivations has not yet been examined.
The aim of this study, therefore, was to characterize the sepA deletion mutant of A. nidulans (sepA4Bm) in submerged cultivation by comparison with the parental strain (A28). Batch cultivations were performed with high (600 r.p.m.) and low (300 r.p.m.) impeller speeds. As the sepA4
Bm strain does not produce septa, this is likely to result in a weaker hyphal ultrastructure and the hyphae are, therefore, more likely to be susceptible to damage caused by shear stresses during cultivation in a bioreactor. Quantitative image analysis was applied to obtain values for the relative size of hyphal elements during cultivations and determine whether fragmentation was more pronounced in the septation deficient mutant. The specific fragmentation rate (qfrag) was also estimated at different stages of growth to further investigate the differences between the two strains. The positioning of nuclei and septa (and thus, compartment lengths) were determined by employing computerized fluorescence image analysis with stains specific for the features of interest. In doing so, we have obtained data on the size of hyphal compartments and distribution of nuclei within both apical and subapical compartments. In addition to obtaining data on the macro- and micro-morphology and the internal organization of hyphae, details of the physiological state of a culture are also of value. In particular, the level of metabolic activity within the culture can be determined by the use of fluorescent probes (Agger et al., 1998
; Cox & Thomas, 1999
) and this adds further detail about the culture.
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METHODS |
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Batch fermentation processes.
Batch fermentations were carried out in a 5 l (working volume) built-in-house fermentation vessel. The reactor consisted of a glass vessel with metal base and top plates through which were fitted a heater, temperature sensor and a cooling unit. The air supply, pH probe and pH control titrants were added through top plate ports. The aeration rate was 1 vol. air (culture vol.)-1 min-1. The temperature was controlled at 30 °C throughout. The pH for inoculation was 3·0, which was increased as the fermentation progressed and was then automatically controlled at 5·0 by addition of 1 M NaOH. Stirrer speed in all processes was initially 100 r.p.m., which was increased as the fermentations progressed to either 300 or 600 r.p.m. The pH and stirrer speed were gradually increased over the time course of the processes to encourage filamentous growth.
The medium for batch fermentations contained (g l-1): glucose, 40; (NH4)2SO4, 15·0; KH2PO4, 3·0; MgSO4.7H2O, 2·0; NaCl, 2·0; CaCl2.2H2O, 0·2; and 1·0 ml l-1 trace element solution containing the following (g l-1): ZnSO4.7H2O, 14·3; CuSO4.5H2O, 2·5; NiCl2.6H2O, 0·5 and FeSO4 . 7H2O, 13·8. Biotin and p-aminobenzioc acid were added aseptically after autoclaving to a final concentration of 1 mg l-1.
Morphological analysis.
Morphological characteristics of the culture were measured using a Leica Quantimet 5501W computer with Leica Qwin software (both from Leica). The program produced digitized images from a Nikon microscope (Optiphot 2) on which was mounted a Kappa CF-8/1 FMC monochrome video camera (Kappa Messtechnik).
Mean area of hyphal elements.
This was determined automatically using a QUIPS routine developed within the Leica Qwin software. Briefly, the threshold level was set on bright field images until the entire area of the hyphae to be measured was detected. The calibration was set such that the projected area (µm2) of hyphal elements was calculated. The mean was computed from measurement of 100 hyphae. All samples were analysed at a total magnification of 160x.
Mean compartment lengths of hyphal elements.
The mean compartment lengths for hyphae were determined for the apical compartment, and first and second compartments distal to this. Lengths were obtained by skeletonizing hyphal trees and using a pre-set calibration to determine the distance between the apex and first septum (apical compartment) and between subsequent septa of each hyphal element (in µm). Mean values were obtained from measurements of 100 hyphae. Samples were analysed at a total magnification of 400x.
For all morphological analyses (including staining protocols), culture medium samples were first diluted to approximately 0·5 g biomass l-1. This prevented the occurrence of overlapping hyphae when the sample was placed on a glass microscope slide.
Estimation of the fragmentation rate.
By assuming that fragmentation occurs with equal probability at any position in the hyphal elements and that the fragmentation function was proportional to the total hyphal length lt, the specific fragmentation rate qfrag(lt,z) (h-1) is given by
![]() | (1) |
(Nielsen & Krabben, 1995 ) (where kfrag(z) is a rate constant (µm-1 h-1). When qfrag is given by (1) a balance for the mean total hyphal length lt,av can be expressed as
![]() | (2) |
(Nielsen & Krabben, 1995 ) where nav is the mean number of actively growing tips and qtip(lt,av,z) is the mean tip-extension rate in the hyphal elements (m tip-1 h-1). If the hyphal diameter is assumed to be constant qtip(lt,av,z) is given by
![]() | (3) |
which, by combination with (2) gives
![]() | (4) |
Thus, the specific rate of fragmentation qfrag(lt,av,z) can be estimated using data on mean projected area of the hyphal elements (which is proportional to the hyphal length if the hyphal diameter is constant), and the specific growth rate µ(z). Since the observed specific growth rate is affected by fragmentation resulting in loss of biomass, the specific fragmentation rate may be underestimated. However, estimation of these rates still enables a quantitative comparison of this complicated process occurring in the different fermentations.
Staining and fluorescent image analysis
Calcofluor white (CFW).
CFW (fluorescent brightener 28, Sigma; 100 µl) (0·3 g l-1) was added to 1 ml diluted sample in a 1·5 ml Eppendorf vial covered with tin foil. Samples were mixed and incubated at room temperature for 5 min. A few drops of the solution were placed on a glass slide and covered with a coverslip for analysis. The dye fluoresces when bound to chitin and glucans, and thus stains cell walls and septa. Images were obtained using fluorescence microscopy on a Nikon Optiphot 2 microscope using a mercury burner and a filter block with 330380 nm excitation wavelength and dichroic mirror with emission >420 nm.
The number of septa per area for each sample was obtained using a QUIPS routine written within Qwin software for image acquisition and analysis. Briefly, this involved automatic detection of the area of the hyphae (as above) using light microscopy. The same field of view was then captured using fluorescence microscopy where septa could be visualized, and the number within the hyphal area counted and entered manually in the QUIPS routine. The mean number of septa per area was determined from 100 fields of view for each sample, viewed at a total magnification of 400x.
Mag fura.
The Mag fura staining method reported by Cox & Thomas (1999) was employed with minor modifications. Mag fura tetra potassium salt (Molecular Probes) was prepared in distilled water to a concentration of 0·1 mg ml-1. Whole medium samples removed from batch fermentations were diluted to approximately 0·5 g l-1 in buffer (citrate buffer, pH 5). Stain (60 µl) was added to 1·5 ml diluted sample, the mixture shaken for 5 s and allowed to stand for 2 min. After this time the supernatant was removed and the sample resuspended in an equal volume of buffer: this was necessary to reduce the background fluorescence of Mag fura, and thus obtain more accurate quantification of the area of stained hyphae. Slides were prepared by pipetting 40 µl stained culture onto a microscope slide, covered with a coverslip. Samples were viewed under fluorescence microscopy using a mercury burner and a filter block with 330380 nm excitation wavelength and dichroic mirror with emission >420 nm.
CFW with DAPI (4,6-diamidino-2-phenylindole dihydrochloride).
The fluorochrome DAPI (Molecular Probes) was used to stain DNA in cell nuclei. Diluted sample (60 µl) was added to 400 µl PBS buffer (pH 9·4) in a 1·5 ml Eppendorf vial coated with tin foil; to this 10 µl DAPI (2 µg ml-1) and 50 µl CFW (0·3 µg ml-1) were added. The vial was shaken gently and allowed to incubate at room temperature for 10 min. A few drops of the solution were then placed on a microscope slide, covered with a coverslip and the sample viewed under fluorescence microscopy as above.
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RESULTS AND DISCUSSION |
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In all processes, maximum mean projected hyphal area was attained towards the end of the phase of rapid exponential growth. Following this, the mean area decreased in all processes (Fig. 1). In the sepA4
Bm strain, however, the decrease in area was far more pronounced than with the parental strain and was most marked in the process where the stirrer speed was high. Additionally, the maximum mean projected hyphal area reached in that cultivation (Fig. 1d
) was lower than when the stirrer speed was low (Fig. 1b
). This and the decline in mean hyphal area from the exponential growth phase onwards, suggests that gross fragmentation was occurring in the cultivations with the sepA4
Bm strain.
The specific fragmentation rate was estimated using data obtained on the mean projected area of hyphal elements and the specific growth rates. The data are presented in Table 1. Early in the processes, the specific fragmentation rate was higher for the parental (A28) strain than the sepA4
Bm strain. On average, sepA4
Bm produces more germ tubes, which are also wider than those of the parental strain (data not shown). Therefore, for the same mean hyphal area in each strain, the mean area of each hypha was lower for the sepA4
Bm strain. Thus, the specific fragmentation rate (qfrag) was estimated to be lower for the sepA4
Bm strain in the early stages of the fermentation. Later in the processes, the specific fragmentation rate of the sepA4
Bm strain exceeds that of the parental strain, most likely due to the fact that the actin cytoskeleton is less organized in this strain (Harris et al., 1999
) and septa (which normally provide a degree of rigidity) are lacking.
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Physiological investigations
The internal organization of the hyphae was investigated by employing specific fluorescent stains and computerized image analysis. It is known that the sepA4Bm strain is deficient in producing septa, at least in the first hours of growth (Momany et al., 1995
).
CFW is a stilbene compound which binds to glucans and chitin in the cell walls and septa of fungi (Agger et al., 1998 ; Kasten, 1993
), and was used in the current study to aid visualization of septa in hyphal samples from batch fermentation processes. The mean projected area of hyphae per septum was calculated for each of the cultivations at a number of time points during the time course. The results are shown in Fig. 3
.
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To explain the observations of Fig. 3, the processes of growth and fragmentation should be considered as these influence the micro-morphology of the culture (Nielsen & Krabben, 1995
). It is reasonable that the maximum area of hyphae supported per septum occurs when the specific growth rate is highest as the apical compartment length has been shown to increase as the specific growth rate increases (Müller et al., 2000
). This may account for the initial rise in area per septum during the exponential growth phase. The area per septum then decreases over the remainder of the time course (Fig. 3
) and this could be due to two distinct processes septation or fragmentation. The most obvious explanation is that more septa would have been produced later in the fermentations, and this coupled with the fact that apical compartment size decreases with a decline in growth rate would have resulted in a lower area per septa being calculated. It is known that the sepA4
Bm strain produces septa later in batch cultivations; however, based on our observations, the number of new septa was low and this alone would not have been sufficient to account for the decreases observed. Additionally, based on results from previous studies (Müller et al., 2000
), the change in the length of the apical compartment could not have resulted in the dramatic decline in area per septum observed in this study. We have already discussed the fact that fragmentation occurs in all cultures during the stationary phase of growth and it is, therefore more likely that this process contributed to the observed decline in the area per septum.
In filamentous fungi, the duplication cycle involves the integration of tip growth, nuclear division, septation and branching (Trinci, 1978 ). When the nuclei have duplicated, they separate and the cycle ends with the laying down of a septum that results in the formation of a new apical compartment (Trinci, 1979
). Tip extension rates (i.e. hyphal duplication) have been shown to increase as the specific growth rate increases (Spohr et al., 1997
; Nielsen & Krabben, 1995
). Information about how a filamentous fungus septates and controls the duplication cycle may be obtained by measurement of the compartment lengths in the hyphae (Müller et al., 2000
). This is of particular interest in the context of the study presented here, where the sepA4
Bm strain is under investigation. Additionally, septation is an important event in the differentiation of fungal hyphae, resulting in compartmentalization of hyphal sections and cellular functions (Smith & Anderson, 1973
). This emphasizes the important role of septation in the life cycle of filamentous fungi, both during their growth in nature and when cultivated in bioreactors.
The lengths of the apical and subapical compartments of the hyphae of both the parental strain and sepA4Bm strains, under both sets of cultivation conditions, are given in Table 2
. Apical compartment length has been shown to increase with increasing specific growth rate in Aspergillus oryzae and Aspergillus niger (Müller et al., 2000
) and in the work presented here, apical compartment length was consistently higher during the exponential phase of growth (26 or 24 h sample) than during the stationary growth phase. The length of the apical compartments was much higher for the sepA4
Bm strain; most hyphae consisted of only one compartment as less septa are formed in this strain. For both strains, apical compartment lengths were lowest during stationary phase when stirrer speed was high; this was likely to be due to the lower specific growth rate (Müller et al., 2000
).
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ACKNOWLEDGEMENTS |
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Received 9 June 2000;
revised 15 August 2000;
accepted 25 September 2000.
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