Laboratoire de Fermentations et de Bioconversions Industrielles1, and Laboratoire des Sciences du Génie Chimique CNRS2, Institut National Polytechnique de Lorraine, 2 Avenue de la Forêt de Haye, BP 172, F-54505 Vandoeuvre-lès-Nancy, France
Author for correspondence: J. L. Goergen. Tel: +33 383 59 58 44. Fax: +33 383 59 57 96. e-mail: Jean-Louis.goergen{at}ensaia.inpl-nancy.fr
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ABSTRACT |
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Keywords: intracellular pH, Streptomyces pristinaespiralis, epifluorescence microscopy, image analysis
Abbreviations: BCECF-AM, 2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, acetoxymethyl ester; pHi, intracellular pH; R527/600, green/red fluorescence ratio (527/600 nm)
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INTRODUCTION |
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Because there has been an ever-increasing demand for antibiotics (new molecules and higher doses) over the last decade (Craig, 1996 ), the optimization of the existing processes and the modification (improvement) of bacterial strains by genetic engineering (Baltz, 1998
) are of great interest. Fine optimization of the pristinamycin production process requires good knowledge of cell physiology during the fermentation. In the context of studies on the physiology of actinomycetes, research teams have focused on metabolic changes occurring when antibiotic biosynthesis takes place under the influence of numerous external factors (Paquet, 1990
; Demain, 1972
). Assuming the existence of a close link between morphology and secondary metabolite production, most reports on image analysis of filamentous micro-organisms, including Streptomyces, present their morphology (Paul & Thomas, 1998
; Cox et al., 1998
; Yang et al., 1996
). Because Streptomyces filaments can have different morphologies (filaments, clumps and pellets), depending on culture conditions and the physiological state of cells, separate studies have been required to investigate the relationship between each morphological type and metabolite production by image analysis (Drouin et al., 1997
; Durant et al., 1994a
, b
; Cox & Thomas, 1992
). In addition, image analysis has been used to determine the location of respiration activity in Streptomyces ambofaciens filaments (Mauss et al., 1997
) and the leakage of cellular components through the membrane of S. ambofaciens using carbon gentian violet staining (Pons et al., 1998
).
Among the parameters controlling metabolic activities, intracellular pH (pHi) plays a major role in the regulation of enzyme activities and transport kinetics of nutrients and metabolites. Furthermore, the pH gradient across cell membranes (pH) is related to cellular energetic mechanisms such as ATP generation (Leyval et al., 1997
; Imai & Ohno, 1995
). pHi assessment in eukaryotic cells is now well documented in the literature; fluorescent probes coupled to spectrofluorometric methods and image analysis techniques have been successfully used to investigate pHi of several cell lines (Heiple & Taylor, 1980
; Slavik, 1983
; Paradiso et al., 1987
; Dix & Verkman, 1990
; Cherlet et al., 1999
). On the other hand, pHi determination in prokaryotic cells by means of flow cytometry (Leyval et al., 1997
) or microscopy coupled to image analysis tools (Siegumfeldt et al., 1999
) remains sparse and pHi assessment in filamentous bacteria, such as S. pristinaespiralis, has not been reported so far. Due to a limited resolution time for distribution measurement of labelled weak acids or bases and NMR methods, other techniques must be considered for such studies. However, because most Streptomyces species tend to grow as pellet and filamentous forms in submerged culture (Whitaker, 1991
), flow cytometry cannot be used. Therefore, epifluorescence microscopy combined with image analysis has emerged as an alternative method.
In this study a protocol was developed for pHi measurement in the pristinamycin-producing species S. pristinaespiralis by epifluorescence microscopy and image analysis using the fluorochrome BCECF-AM [2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, acetoxymethyl ester]. A second consideration concerns the methodology applied to monitor the variations of pHi during pristinamycin-producing batch culture of S. pristinaespiralis.
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METHODS |
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Media.
The following media were used in this study. Complex medium (g l-1): sucrose, 15; corn steep (Roquette), 5; (NH4)2SO4, 10; K2HPO4, 1; NaCl, 3; MgSO4.7H2O, 0·2; CaCO3, 1·25; pH 6·8; sterilized for 20 min at 120 °C. Synthetic medium (g l-1): glucose, 30; malonic acid, 7·5; L-arginine, 3; L-glutamate, 1·5; K2HPO4, 0·75; MgSO4.7H2O, 0·3; FeSO4.7H2O, 15; ZnSO4.7H2O, 0·55; CaCl2.2H2O, 0·4; 3-morpholinopropanesulfonic acid, 20; pH 6·8; sterilized for 20 min at 110 °C. K2HPO4 and CaCl2 in concentrated solutions were sterilized by filtration on 0·22 µm filters (Millipore) and added separately to avoid precipitation. All chemicals were analytical grade.
Culture conditions.
Inocula were prepared by pouring 0·8 ml of a thawed spore solution calibrated at 3 x 108 c.f.u. l-1 into 500 ml baffled shake flasks containing 80 ml complex medium. Flasks were shaken at 250 r.p.m. and 28 °C for 44 h using an orbital shaker (Ika).
Bacteria used during the experiments dedicated to method development were grown in 300 ml Erlenmeyer flasks, whereas monitoring of pHi of pristinamycin-producing S. pristinaespiralis was performed on cultures grown in a 2 l bioreactor (CMF 100; Alpha-Laval-Chemap). Inocula (2 ml) were poured into 250 ml Erlenmeyer flasks containing 30 ml synthetic medium. Cells were grown with agitation at 250 r.p.m. and 28 °C. For bioreactor batch cultures, 80 ml preculture was inoculated into 2 l synthetic medium at 28 °C. The oxygen level was maintained at a minimum of 30% of air saturation and controlled through stirring conditions. Agitation with Rushton impellers ranged between 300 and 1500 r.p.m.
Cell staining.
All experiments were carried out at 4 °C in a dark room. Stock solutions (50 µl samples) of 1 mM BCECF-AM (Molecular Probes) in DMSO were stored in the dark at -20 °C. Culture samples were directly diluted in PBS to obtain a final OD660 of 1·0. Cells were incubated at 28 °C on a rotary shaker with different concentrations of BCECF-AM (2·550 µM) for incubation times ranging from 0 to 40 min.
In vivo calibration.
The [H+-K+] carboxylic ionophore nigericin and neutral ionophore valinomycin (Sigma) were used for pHi calibration as described elsewhere (Musgrove et al., 1986 ; Pressman, 1976
). Nigericin was dissolved in absolute ethanol and valinomycin in DMSO at final concentrations of 1 and 10 mM, respectively. Both solutions were stored at -20 °C. The cells used for the establishment of calibration curves were stained as described above. After a 30 s centrifugation at 6000 g and the removal of supernatant, cells were resuspended in high [K+] buffers at different pH values (6·58·5). High [K+] buffers were obtained by mixing appropriate quantities of 135 mM KH2PO4/20 mM NaOH and 110 mM K2HPO4/20 mM NaOH. Buffer solutions were filtered before use through 0·22 µM filters and stored at 4 °C. When stated, osmolality of each buffered solution (measured with a Roebling osmometer) was adjusted to that of the fermentation broth by adding xylose. Nigericin was added at a final concentration ranging from 5 to 40 µM for 10 min. Valinomycin was added simultaneously to 20 µM nigericin at concentrations varying from 0 to 10 µM for different incubation times (530 min). Cells were then centrifuged again as described above, resuspended in the same high [K+] buffer and kept on ice.
Double emission ratio technique.
An epifluorescent microscope (DMRB; Leica Leitz) with x10 magnification and a 0·30 numerical aperture PL Fluotar objective was used. The final magnification was x100, because of the camera and the x10 magnification of the objective. The light source was a 50 W Hg lamp (Osram). Excitation and emission band pass filters were assembled. Whereas the excitation filter was the same for both modules (480/40 nm), two different emission filters were used: 527/30 nm (green) and 600/40 nm (red). Stained cells (20 µl) were carefully spread onto a glass slide. After the 527 nm image (green image) was taken and saved, another image of the same field (same pellet) was acquired at 600 nm (red image). Photobleaching of the stained samples was considered as negligible because only a 5% decrease in fluorescence intensity was registered after five ratio measurements on the same field (10 successive excitations). Autofluorescence of the cells was shown to have no influence on the calculated ratio, since the mean fluorescence ratio at 527/600 nm (R527/600) was 1·00 (SD<0·01) and pixel level intensity was inferior to image processing threshold values. No correlation could be found between ratio values and pellet size, suggesting that the methodology can be used for pellets of different sizes. For each experiment, 2030 images were recorded at each emission wavelength. Error bars on the graphs represent the SD of mean R527/600 values.
Image processing.
An integrated controlled CCD monochrome camera (Cohu) was fitted onto the epifluorescence microscope. It was controlled via a PC through an acquisition card (Secad Vision) which allowed capture of 768x576 pixel TIFF images coded on 256 grey levels. The integration time was set to 25 ms for each image. Image treatment was carried out on a PC using professional image analysis software (Visilog; Noesis).
The aim was to obtain images with pixel values directly connected to the pHi value (pH image) by using the double ratio technique described. The first part of the treatment deals with the segmentation of these images. This operation was carried out by an automatic thresholding technique based on image entropy. Concerning the image, entropy is maximum for an equally distributed grey level histogram and equal to zero for an image with only one grey level. This technique is used to segment images into two regions by maximizing the total entropy (Coster & Chermant, 1989 ). It has been shown that such a segmentation best fits images containing pellets (Pons & Vivier, 1999
). Consequently, two binary images were obtained, one for each initial grey level image. The binary image only contains the relevant information (object), with pixels set to 1 and background pixels set to 0. Then, a logical operation (intersection) between these images gives a single binary image representing the common part of the initial images. After morphological operations (dilation and reconstruction) to eliminate artefacts due to the thresholding, a final binary image is obtained and used as a mask for further operations. Masking is a point operation between a grey level and a binary image that produces a grey level image. In such an image, only pixel parts of the object (i.e. the pellet) are visible in grey level, the background pixels being set to black (0). Then, both grey level images (527 and 600 nm) were masked and the mean grey levels of the pellets and the SD values were computed. These values are good indicators of the quality of calibration images and they were used as criteria for the final pH calculation. Finally, after transformation in floating point images to allow arithmetic operations, a pixel to pixel division between images was performed to get the pH image. Mean grey level and SD values were also calculated on this final image to give values related to pHi.
Additional analytical methods.
During batch fermentations samples were collected to determine glucose, pristinamycin and dry weight biomass levels.
OD660 was measured according to the method of Lubbe et al. (1985) by means of a Beckman spectrophotometer, DU 7500.
Dry weight biomass was determined gravimetrically by filtering 10 ml fermentation broth on pre-weighed 0·45 µm cellulose acetate membranes (Sartorius). Membranes were washed twice with 0·85% NaCl and placed in an oven at 100 °C for 12 h before being weighed again. An OD660 vs dry weight correlation curve allowed dry weight biomass determination.
Pristinamycins were extracted and analysed by HPLC as described by Thibaut et al. (1995) with a Spectra-Physics HPLC system.
Glucose concentration was determined by HPLC according to the protocol described by Rondags et al. (1998) .
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RESULTS |
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Because magnifications of x1000 and x400 led to a dramatic photobleaching of the fluorescent probe, a final magnification of x100 was chosen. Using this magnification, a 25 ms exposure time was used to capture images.
Optimization of the BCECF-AM staining technique.
The protocol for pHi measurement was first optimized by testing the effect of various dye concentrations and staining times on the measured fluorescence of S. pristinaespiralis. Cell samples were submitted to increasing concentrations of BCECF-AM, from 1 to 60 µM, for 30 min at 28 °C. The measured mean R527/600 values of the samples are reported in Fig. 1.
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To optimize the ionophore incubation time, suspensions of S. pristinaespiralis were stained with BCECF-AM and incubated with 20 µM nigericin/1 µM valinomycin at 28 °C in high [K+] buffer (pH 7·0) for times varying from 10 to 30 min. R527/600 was constant (1·46±0·05) whatever incubation time was used. On this basis, a 10 min incubation time was chosen for further experiments.
For the calibration of the technique, cells were resuspended in high [K+] buffers at different pH values ranging from 6·5 to 8·5. The osmolality of the buffers was adjusted to that of the culture medium by means of xylose to avoid osmotic shock. Nigericin and valinomycin were added to the samples to equilibrate the pHi of the stained cells to the pH of the surrounding buffer.
Calibration curves green/red fluorescence ratios vs pH were established for three samples of growing cells sampled after 24 h culture (Fig. 4). After being captured by the monochrome camera, image analysis showed that the equation of the calibration curve was slightly different for pellets with grey level images between 20 and 25, and between 25 and 30, at 600 nm. Thus, two calibration curves were used. One curve was drawn for pellets having mean grey levels between 20 and 25 at 600 nm and another was determined for mean grey levels between 25 and 30 (Fig. 5
). For pellets with grey levels at 25 at 600 nm, pHi values were calculated using both equations.
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To investigate a possible effect of the three-dimensional size and shape of the pellets on axial resolution (depth of field), topographic sections across pH pellet images were taken (Fig. 6a). If there was an effect, a central pixel displaying a pHi similar to another at the periphery of the pellet would have a higher R527/600. In fact, as shown in Fig. 6(b)
, the mean R527/600 values of the peripheral pixels (at the termini of the straight line) were identical to the values of the pixels from the centre of the pellet (middle of the line), indicating that the surface fluorescence is not affected by the depth of the pellet. Pellets presented a homogeneous pHi over a large part of their surface and the SD of mean R527/600 values for pixels of a given pHi remained very low over the whole surface of the pellets.
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Bioreactor batch culture of pristinamycin-producing S. pristinaespiralis
The protocol presented for pHi determination was applied to the pristinamycin-producing S. pristinaespiralis grown under stable pH conditions (external pH 6·8). To evaluate growth, glucose consumption, pristinamycin production and pHi of S. pristinaespiralis, a bioreactor was operated in batch mode over a 40 h period (Fig. 7a, b
). The pHi of the pellets of each culture sample was calculated with the calibration curve corresponding to the mean grey level at 600 nm. The dry weight biomass level, initially 0·4 g l-1, increased to reach a maximum of 8·0 g l-1 within 40 h (Fig. 7a
). Growth stopped independently of carbon limitation as the glucose concentration was still around 20 g l-1 at that time.
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This culture was performed in triplicate (data not shown) and identical pHi profiles were observed, though minor differences regarding the pHi values were obtained during the three processes (± 0·4 pH units).
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DISCUSSION |
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The fluorochrome BCECF-AM, with a pKa of 7·0, was found to be efficient for pHi determination of neutrophilic bacteria, such as S. pristinaespiralis, grown in a bioreactor: the fluorescence was proportional to the pHi of the cells between pH values of 6·5 and 8·5, and the fluorescence of the stained cells was stable for at least 30 min.
During this study only pellets of S. pristinaespiralis were taken into account because the bacteria mainly grow in this form during the pristinamycin-producing process. Another reason for this choice is that the origin of dispersed filaments present in the culture medium is not always clear. They can be present as a consequence of the alteration of fluffy pellets caused by shearing forces produced by the increased agitation necessary for the regulation of dissolved oxygen. Moreover, simultaneous analysis of all morphologies present in the culture has been reported to be impossible since a x100 oil immersion objective (x1000 final magnification) generally requires the use of coverslips for filament analysis which can disturb the physiology of the cells in the pellets (Nielsen et al., 1995 ; Cox et al., 1998
).
The pHi of S. pristinaespiralis cells only from the surface of the pellets (projected area) was considered. In fact, surface cells within the pellets can be considered as the most active cells because pellets have been described to have hollows in their centre (Thomas, 1992 ). These hollows form due to cell necrosis as a result of limitations in oxygen and nutrients.
In vivo calibration is essential to avoid response variability due to differences in microscope focusing between BCECF solutions (in vitro calibration) and BCECF-AM-stained pellets.
Satisfactory correlation coefficients and linearity were found with the proposed nigericin-valinomycin protocol over the pH 6·58·5 range. Contrary to the results of Boyarsky et al. (1996a , b
) using eukaryotes, enough K+ was able enter the pellets to equilibrate extracellular and pHi, even at basic pH values. Addition of xylose, resulting in increased osmolality of high [K+] buffers, could be used to avoid K+ efflux in response to hypotonic shock. Such inward or outward cationic fluxes have been described in response to medium osmolality changes (Guillouet, 1996
). One explanation for the efficient combination of valinomycin and nigericin at these concentrations could be their overwhelming action on mechanisms of cell regulation, allowing equal distribution of K+ at intracellular and extracellular levels, without any residual K+ gradient, contrary to the results of Boyarsky et al. (1996a
, b
).
Slight differences in 600 nm pixel intensity for images of pellets resuspended in buffer with the same pH could generate slightly different R527/600 ratios, as seen during this study. Differences in staining have been reported for cells having different esterase activities in the culture (Franck et al., 1996 ). However, the difference in 527 nm fluorescence should have been of the same order. One explanation could be a different sensitivity of the camera, which is described as more efficient around 527 nm, at the two wavelengths. Another reason is that 600/40 nm fluorescence is not really an isosbestic point (Franck et al., 1996
). Actually, this artefact could be corrected using two different calibration curves, according to the level of fluorescence at 600 nm.
The advantage of the image analysis algorithm developed here is that it does not modify any pixel level. This results in the collection of unmodified data and it gives a more accurate estimation of actual pHi. Since fluorescence was pellet-area-independent, contrary to other studies dealing with morphology by image analysis (Sieracki et al., 1989 ; Durant et al., 1994a
, b
), no delimitation problems were encountered by using an automated entropy threshold. The simplicity and rapidity (less than 1 min for calculation) of this procedure are favourable to on-line routine analysis during fermentation processes.
This paper presents for the first time a method for the determination of pHi of filamentous bacteria by means of epifluorescence microscopy and image analysis using a pH-sensitive fluorescent probe. Although a positive control was not employed during this study (use of a bacterium with a known pHi), the different tools and techniques utilized have already been reported to be efficient for the determination of pHi in bacteria. First, BCECF is a valuable fluorochrome to determine pHi since it has been shown to perfectly corroborate 31P-NMR spectroscopy data obtained for Propionibacterium acnes (Futsaether et al., 1993 ). Moreover, Slavik (1997)
has shown that the BCECF emission ratio technique can be employed for confocal microscopy to determine pH. In addition, pHi determination of Listeria innocua using epifluorescence microscopy and image analysis (Siegumfeldt et al., 1999
) confirmed the results obtained by spectrofluorimetry (Breeuwer et al., 1996
).
During this study, the pHi of S. pristinaespiralis grown in batch culture was measured by an image analysis-based method using the fluorochrome BCECF-AM at a final concentration of 20 µM at 28 °C and for 30 min. As shown, pHi could be correlated to pristinamycin excretion and glucose consumption in batch process. In fact, pHi was found to be a good marker of the physiological state of the cells cultivated under pristinamycin-producing conditions. Furthermore, pHi assessment can contribute to the understanding of variations in carbon fluxes through the enzymes involved in glucose metabolism, and pH calculation may help in the establishment of kinetic models for excretion and consumption of metabolites. Finally, the new method presented in this paper for pHi assessment of S. pristinaespiralis could be extended to filamentous bacteria of fungal cells cultivated in vitro, provided that the staining protocol was adapted to each cell line.
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ACKNOWLEDGEMENTS |
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Received 14 March 2000;
revised 19 June 2000;
accepted 12 July 2000.