Molecular Microbial Ecology Group, Department of Microbiology, Technical University of Denmark, Building 301, DK-2800 Lyngby, Denmark1
Biofilm Imaging Facility, Center for Environmental Biotechnology, University of Tennessee, Knoxville, TN 37932-2575, USA2
Author for correspondence: Søren Molin. Tel: +45 4525 2513. Fax: +45 4588 7328. e-mail: soeren.molin{at}biocentrum.dtu.dk
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ABSTRACT |
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Keywords: Gram-positive bacteria, biofilms, green fluorescent protein tagging, anaerobic growth
Abbreviations: GFP, green fluorescent protein
a Present address: National Institute for Dental Craniofacial Research, NIH Oral Infection and Immunity Branch, Bldg 30, 30 Convent Drive, Bethesda, MD 20892, USA.
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INTRODUCTION |
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The GFP fluorophore has been characterized in detail. It consists of a p-hydroxybenzylideneimidazolinone formed autocatalytically by cyclization and oxidation of the Ser-Tyr-Gly sequence at positions 6567 with a time constant of approximately 24 h at 22 °C and atmospheric pO2 (Heim et al., 1994 , 1995
). The final step in protein maturation is dehydrogenation by molecular oxygen of residue 66 (Cubitt et al., 1995
; Heim et al., 1994
; Reid & Flynn, 1997
). In order to enhance the fluorescence of GFP, to broaden the useful pH and temperature ranges of the protein, to increase the rate of fluorophore formation, and to shift wavelengths of excitation and emission, several mutations of the protein have been introduced (Cormack et al., 1996
; Crameri et al., 1996
; Delagrave et al., 1995
; Ehrig et al., 1995
; Elsliger et al., 1999
; Heim et al., 1994
; Heim & Tsien, 1996
; Kimata et al., 1997
; Patterson et al., 1997
; Siemering et al., 1996
; Ward, 1998
). One variant (Gfpmut3b) with two mutations in the chromophore region (S65G, S72A) fluoresces 21 times more intensely than wtGfp, and was observed to mature with a time constant of approximately 45 min (Cormack et al., 1996
). Throughout the present study, a variant of this protein (Gfpmut3*) was used, which has an additional mutation at position 2 (S2R) that permits introduction of a SphI site in the gene (Andersen et al., 1998
).
Many Gram-positive bacteria, which are interesting in relation to industrial applications or as human and animal pathogens, are fermentative and microaerophilic. It has generally been assumed that GFP was less useful as a reporter in these organisms because of the requirement for oxygen in fluorophore formation (Cody et al., 1993 ; Cubitt et al., 1995
; Heim et al., 1994
; Prasher et al., 1992
; Reid & Flynn, 1997
). In addition, there are fewer handy genetic tools available for Gram-positive bacteria compared to those designed for enterics and other Proteobacteria. It is therefore understandable that the current literature concerning GFP as a reporter in, for example, streptococci and related bacteria is quite limited. Besides the oxygen requirements, the actual rate of oxidation, and the stability of GFP under different conditions, are uncertain (Cormack et al., 1996
; Heim et al., 1994
; Reid & Flynn, 1997
). The design of standardized GFP test plasmids (a broad-host-range shuttle vector carrying the gfp gene under control of a strong constitutive promoter) and clarification of the limitations of the specific growth conditions of these organisms in relation to obtainable fluorescence signals are therefore warranted.
The Gram-positive Streptococcus gordonii is one of many bacterial species involved in the formation of dental plaque. In the oral environment, S. gordonii is an initial colonizer, which grows attached to the tooth surface (Kolenbrander & London, 1993 ). It is important to characterize the general performance of this bacterium when growing in biofilms in addition to what has been investigated for cells in the planktonic state, since sessile populations represent in vivo conditions more accurately than planktonic bacteria. Cultivation of bacteria in flow-chambers makes it possible to study biofilm communities, and this model system approach has proven to be much more representative of conditions in the oral cavity than are static systems (Palmer & Caldwell, 1995
).
The aims of this study were (i) to design and characterize genetic tools targeting Gram-positive bacteria in general and microaerophilic species in particular; (ii) to test the limits of the use of GFP as a marker under suboptimal maturation conditions (low pH, low O2), and thereby to address these limitations in a practical application of GFP the use of the gfpmut3* gene (Andersen et al., 1998 ) as a molecular marker in the Gram-positive bacterium S. gordonii DL1 grown in batch cultures and in biofilms under aerobic and anaerobic conditions; and (iii) to determine which growth conditions allow sufficient GFP reporter signals to be useful in connection with in vitro as well as in situ studies.
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METHODS |
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Flowcell system.
S. gordonii DL1 was grown as biofilms in glass flowcells (channel dimensions 3 mm wide and 1 mm high) for microscopy (Palmer & Caldwell, 1995 ). Medium for the flowcells was supplied from a reservoir through silicone tubing. A peristaltic pump (Watson Marlow set at rate 12 ml h-1) pulled medium through the system and led the effluent to a waste container.
Glove bag.
In order to obtain anaerobic conditions for the flowcells, an Inflatable Glove Bag (model X-17-17, Instruments for Research and Industry, Cheltenham, PA, USA) was used to contain the experimental set-up (Hansen et al., 2000 ). The gas environment in the glove bag was 95:5 N2/CO2.
Measurement of optical density.
A Shimadzu spectrophotometer model UV-1201 (SpectraChrom, Brøndby, Denmark), was used for measurements of OD600.
Measurement of pH.
A Sentron pH meter model 1001 was used for measurements of pH. Calibration was performed using pH 4·0 and pH 7·0 buffers.
Measurement of dissolved oxygen.
The concentration of dissolved oxygen in the anaerobic medium in anaerobic culture tubes (Bellco) and in the reduced flowcell medium was determined colorimetrically using CHEMets Dissolved Oxygen Ampoules (CHEMetrics, Calverton, VA, USA). All measurements were done in the glove bag. Tubed anaerobic medium without L-cysteine contained 0·1 p.p.m. dissolved oxygen. Medium with L-cysteine for the flowcells contained 0·025 p.p.m. dissolved oxygen (limit of detection).
Scanning confocal laser microscopy.
Flowcells were removed from the glove bag prior to microscopic examination. In the shift experiment from anaerobic to aerobic medium, the medium supply was changed as soon as the flowcell was removed from the glove bag, and the biofilms were immediately observed using a Leica TCS-NT confocal laser microscope. The microscope settings were as follows: excitation at 488 nm, emission at 530/30 BP into channel 1 to record GFP fluorescence, 100x1·4 NA oil-immersion lens at an Airy disc setting of 0·9, 1 µm steps collected with four frames averaged at each step. When an increase in fluorescence intensity was expected, the PMT gain was deliberately reduced in order to be able to keep this setting throughout the entire experiment and still avoid saturation in the brightest images. Biofilms were also subjected to microscopic examination after 18 h and after 2 d of growth.
Epifluorescence microscopy.
Fluorescence in suspended cells from liquid cultures was observed by applying 4 µl of culture on a microscope slide followed by examination using either the confocal laser microscope as described or a Carl Zeiss Axioplan epifluorescence microscope. For the latter, the excitation source was a 100 W HBO bulb, and digital images were captured with a 12-bit cooled slow-scan charge-coupled-device camera (KAF 1400 chip; Photometrics). The charge-coupled-device camera was controlled by the PMIS software (Photometrics), and an FITC filter set was used for the excitation and detection of GFP.
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RESULTS AND DISCUSSION |
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Expression of GFP in batch cultures of S. gordonii DL1(pCM18)
The CAMG broth medium used for growth of S. gordonii DL1 was prepared either anaerobically or aerobically. The conventionally prepared anaerobic medium contained 0·1 p.p.m. (0·1 mg l-1, approximately 3 µM) dissolved oxygen. There have been several reports on the oxygen requirements of GFP for the final oxidation of the mature, cyclized chromophore (Cody et al., 1993 ; Heim et al., 1994
, 1995
; Inouye & Tsuji, 1994
; Prasher et al., 1992
), and describing the kinetics of the steps involved in chromophore formation including the final oxidation of the tripeptide (Reid & Flynn, 1997
). We found that exponentially growing cells were fluorescent in anaerobic medium, i.e. in the presence of 0·1 p.p.m. dissolved oxygen, even after several transfers to secure sufficient dilution of the initial inoculum (data not shown). This implies that a significant proportion of GFP was oxidized by the trace amounts of oxygen found in our anaerobic pressure tubes. Several applications of GFP have faced troublesome interpretations of the fluorescence due to potentially limiting concentrations of oxygen, e.g. cells in the centre of microcolonies in otherwise aerobic environments. The present demonstration that very low amounts of oxygen are needed for maturation of GFP opens the possibility of using this reporter under conditions of near-anaerobiosis.
S. gordonii is a homofermentative bacterium: glucose is fermented to twice the equimolar amount of lactate. The production of acid reduces pH in the medium as lactate accumulates. As shown in the initial characterization of GFP (Prasher et al., 1992 ), the fluorescence of the protein is significantly influenced by changes in pH. Elsliger et al. (1999)
argued that the quenching of fluorescence at low pH is due to the formation of a protonated form of the chromophore phenolate in the GFP S65T variant. This pH sensitivity of GFP was exploited by Robey et al. (1998)
and Miesenböck et al. (1998)
, using mutants of GFP as in vivo reporters of cytoplasmic pH. The pH sensitivity becomes problematic when GFP is used in lactic acid bacteria in batch cultures. For several lactic acid bacteria, it has been found that the intracellular pH decreased with the extracellular pH, maintaining a constant
pH of 0·50·8 units (Cook & Russell, 1994
; Siegumfeldt et al., 2000
). Similarly, epifluorescence microscopy of single cells from an aerobic exponentially growing culture of S. gordonii DL1 showed that fluorescence was equally distributed among the cells in the population. As pH decreased in the medium, fluorescence intensity decreased. If the growth medium is supplemented with different amounts of glucose (0·2 and 0·5%), the cultures reach stationary phase at different pH values. This makes it possible to compare the pH effect unbiased by the change in growth phase. In medium with 0·2% glucose, the pH decreased from 7 to 6, resulting in a corresponding drop in fluorescence intensity. However, when the medium was supplemented with 0·5% glucose, the pH decreased to 4·5, with a further decrease in fluorescence. Fluorescence from cells at or below about pH 5·5 was only detectable through a strong amplification of the signal, making the fluorescence practically useless, as the cells lost edge definition (they were difficult to spot). These data are summarized in Fig. 2
. For each sample point, a set of epifluorescence micrographs was obtained, and the fluorescence intensity was estimated using the maximum pixel intensity option from the PMIS (Photometrics) software. Although streptococci to some extent may control their internal pH (Cook & Russell, 1994
), Fig. 2
clearly shows the effect of external pH on GFP fluorescence in vivo. This phenotype was independent of oxygen supplied by vigorous shaking of the culture.
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Expression of GFP in biofilms
As in batch cultures, green fluorescence in biofilms was observed in medium containing approximately 0·1 p.p.m. oxygen. The pH in the effluent from a biofilm grown for 2 d was approximately 7 (data not shown). GFP fluorescence in aerobic biofilms of S. gordonii DL1(pCM18) was homogeneously distributed throughout the biofilm until a very thick cell-layer (>50 µm) quenched the fluorescence signal. The continuous flow of medium through the flowcell system prevents accumulation of lactic acid and a resultant drop in pH, and consequently the GFP fluorescence signal intensity allowed excellent recordings of the cells by scanning confocal laser microscopy throughout the course of the biofilm development (up to 4 d).
When the entire flowcell set-up was placed in a glovebag as described by Hansen et al. (2000) , and L-cysteine was added to the medium, the biofilms could be maintained under strictly anaerobic conditions (
0·025 p.p.m. oxygen, equivalent to
0·8 µM). Under these conditions the biofilms increased in biomass faster than parallel aerobic biofilms, but similar biofilm characteristics (distribution of biomass and cellular morphologies) were developed under the two sets of conditions. However, green fluorescence was not observed in these biofilms until after a shift to aerobic medium. Inspection of the fluorescence development in a shift from anaerobiosis to aerobiosis using the scanning confocal microscope showed that fluorescence was detectable 4 min after oxygen was supplied (Fig. 3
). During the next 16 min, a further increase in fluorescence intensity occurred, but after an additional 20, 40, 60 and 100 min, little or no increase in fluorescence could be observed. In Fig. 3
, single optical sections directly at the substratum show the fluorescence increase at the deepest regions of the biofilm (in the middle of microcolonies) as well as in cells exposed to pore-space fluid (cells at the edge of those microcolonies), and are displayed in conjunction with images of the entire biofilm biomass (maximum projections).These micrographs of single sections indicate the uniform increase of fluorescence in this biofilm as do the side panels (zx and zy scans) on the entire stack of images. Neither vertical nor horizontal gradients of fluorescence through the biofilm were observed fluorescence increased the same way in cells from the middle of a micro-colony as in those on the surface, indicating a rapid distribution of oxygen through the flowcell and through the S. gordonii biomass.
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The actual in vivo requirements of oxygen for GFP fluorescence under limiting oxygen tensions have been determined. Maturation of the GFP fluorophore occurs at levels of oxygen as low as 0·1 p.p.m., whereas no fluorescence was detectable at 0·025 p.p.m. oxygen. This implies that the use of GFP is not restricted to aerobic bacteria and well-aerated systems microaerophilic organisms may also be characterized using GFP. Oxygen-dependent maturation is very fast, since a fully fluorescent biofilm was obtained in less than 20 min. A low pH in the environment results in low fluorescence yield, and for organisms which most often grow fermentatively it is highly important to control external pH.
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ACKNOWLEDGEMENTS |
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Received 2 November 2000;
revised 21 December 2000;
accepted 23 January 2001.