School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK1
Author for correspondence: Lynne E. Macaskie. Tel: +44 121 414 5889. Fax: +44 121 414 5925. e-mail: L. E.Macaskie{at}bham.ac.uk
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ABSTRACT |
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Keywords: attached cells, starvation, phosphohydrolase, confocal laser scanning microscopy, fimbriae
Abbreviations: CLSM, confocal laser scanning microscopy; EPS, extracellular polymeric substance(s); SEM, scanning electron microscopy; TEM, transmission electron microscopy
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INTRODUCTION |
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Understanding of the processes of biofilm formation remains incomplete. Factors such as the hydrodynamics of the bulk fluid (Kugaprasatham et al., 1992 ; Peyton, 1996
), the nature of the substratum (Dalton et al., 1994
; van Loosdrecht et al., 1995
), species composition (Lawrence et al., 1991
) and nutrient availability (Huang et al., 1994
; Ohashi et al., 1995
; Peyton, 1996
) influence biofilm formation, but it is still not clear exactly how these factors interplay, or which factors dominate. Therefore, the primary aim of this study was to determine the extent to which a single change in growth conditions affects the formation of a biotechnologically useful monospecies biofilm, excluding possible variables such as interspecies interactions and communication which are often observed (James et al., 1995
; Davies et al., 1998
).
The test organism, a Citrobacter sp. (NCIMB 40259) has been used for the bioremediation of heavy metals via the activity of an acid-type phosphatase enzyme (Jeong & Macaskie, 1999 ) which liberates
from a suitable organic phosphate donor with the stoichiometric precipitation of metal cations (M2+) as insoluble MHPO4 at the cell surface (Macaskie et al., 1995
; Finlay et al., 1999
). Conditions promoting maximum biofilm growth may not necessarily produce maximum enzymic activity. Therefore phosphatase specific activity was investigated in parallel with morphological and quantitative studies of the biofilm with respect to the effect of nutrient limitation. The latter is known to promote changes in cell physiology and composition (Harder & Dijkhuizen, 1983
; Breedveld et al., 1995
), but the consequences of these changes on biofilm formation and structure are largely unknown. Carbon limitation can promote cell adhesion to surfaces (Ellwood et al., 1982
), while the C:N ratio of the medium can affect species composition (Ohashi et al., 1995
), plasmid stability and the ratio of polysaccharide to protein content within biofilms (Haung et al., 1994
; Ohashi et al., 1995
). Upregulated alkaline phosphatase was visualized in phosphorus-limited biofilms (Huang et al., 1998
) but in contrast acid phosphatase production is regulated by the carbon status of the medium and the enzyme has a less clearly defined role (Jeong & Macaskie, 1999
; Macaskie et al., 2000
). In this study Citrobacter sp. strain NCIMB 40259 was grown in a chemostat under carbon, phosphorus or nitrogen limitation and the phosphatase activity, cellular morphology and biofilm formation and structure were evaluated. The results are discussed with respect to factors affecting biofilm formation.
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METHODS |
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Assay of phosphatase activity.
Phosphatase specific activity was measured by the release of p-nitrophenol (PNP) from p-nitrophenyl phosphate as described previously (Macaskie et al., 1995 , 2000
; Jeong et al., 1997
; Finlay et al., 1999
; Bonthrone et al., 2000
). One unit is defined as 1 nmol PNP released min-1 (mg bacterial protein)-1 with the protein concentration measured using the CuSO4/bicinchoninic acid method (Sigma protein test kit TPR0562) and bovine serum albumin as the standard. For estimation of biofilm enzyme activity, samples were prepared by washing and resuspending the biofilm in isotonic saline and vortex-mixing (4 min) to break apart cell aggregates prior to assay.
Electron microscopy.
Each sample was fixed immediately in 2·5% (v/v) glutaraldehyde in 0·1 M sodium cacodylate buffer (pH 7·2, 4 °C, overnight). Dehydration was in a graded ethanol series, followed by critical-point drying with carbon dioxide. Samples were sputter-coated with gold prior to examination using a scanning electron microscope (Hitachi 2300; accelerating voltage 15 kV). Samples for negative staining for transmission electron microscopy (TEM) were washed three times in isotonic saline. The cell density of the sample was adjusted to OD600 0·2. The samples were attached to a carbon-coated copper grid by air fixation, stained with 10 µl sodium phosphotungstate solution (5%, w/v, pH 7·3; 5 min), washed by brief dipping in distilled water (subsequently drawn off onto filter paper) and dried for 1 min under a lamp. Samples were viewed using a JEOL 1200 EX electron microscope (accelerating voltage 80 kV).
Confocal laser scanning microscopy (CLSM).
Biofilm was removed from one side of cover slips by washing with 70% (v/v) ethanol. The silicone tubing was removed and the biofilm on the other side was stained with 0·005% (w/v) acridine orange for 5 min in the dark and washed three times in sterile isotonic saline. The biofilm was viewed under a Bio-Rad MRC 600 CLSM attached to a Nikon Diaphot microscope and an appropriate set of filters (488 nm excitation filter, 510 nm longpass filter and <515 nm barrier filter). The alignments of the laser and monitor set-up were checked according to the manufacturers instructions. The sample was observed under a x40 (dry: NA 0·75) andx60 (oil immersion: NA 1·4) lens, with the latter used for image acquisition. Use of an inverted microscope (sample viewed from the clean side of the cover slip, under oil), circumvented the need for a cover slip over the film, which would have compressed the biofilm structure. Five orthogonal (vertical) section images were taken at random from each sample using the Vertical Section function of the instrument and saved onto a Zip disk (Iomega). Height measurements were made from these transects, with each measurement taken vertically from the cover slip to the top of the biofilm. Twenty measurements were taken at regular distances (8·3 µm apart) across the horizontal transects. Measurements, calibrated against fluorescent beads with a diameter of 15 µm (Molecular Probes), were made using COMOS software as supplied. The area fraction (% coverage) was measured from a series of optical sections imaged at different depths at the same xy coordinates (horizontal planes at different depths: z series). Measurements were made using the BAND function of the COMOS software. The threshold value was set manually and retained for all sections in the same plane.
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RESULTS |
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Biofilm formation in nutrient-limited continuous culture
Biofilm was apparent on the vessel walls after 96 h under lactose limitation, but not under N or P limitation. Table 2 confirms that the wet weight of biofilm harvested from the known surface area of the reactor was significantly higher (F2,6=101; P<0·01) for lactose-limited cultures. The dry weight of biofilm was estimated in parallel for each experiment; the wet weight/dry weight ratio was 10:1, in accordance with previous studies using batch-cells (P. Yong & L. E. Macaskie unpublished). The carbon source was also important; glucose limitation produced a mean biofilm biomass of 0·31 g, which was only 10% of that under lactose limitation (3·2 g) and was comparable to P-limited cultures (Table 2
). In an extended experiment a culture grown under N limitation (with lactose) for 15 d gave 0·47 g of biofilm biomass, i.e. the amount of biofilm had more than doubled, but was still 86% less than for lactose-limited cells.
Examination of surface features of biofilm and planktonic cells
Cells were negatively stained and examined by TEM. A minimum of 50 cells was observed from each sample. Almost all biofilm cells (91%) from the lactose-limited medium had numerous appendages (fimbriae: Sharon, 1984 ) covering the whole cell surface (Fig. 2a
) but only 55% of planktonic cells from the same chemostat displayed these. Only a minority of either type of cells grown under P, N or glucose limitation (<30%) showed appendages. Each cell displayed a large number of fimbriae or none (Fig. 2a
, b
); appendages were divisible into clearly visible large structures and an indistinct fuzzy layer covering the cell (Fig. 2c
), probably comprising curli (Olsen et al., 1989
; Fig. 2c
, inset).
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DISCUSSION |
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Substantial biofilm growth was observed under lactose (this study) or glycerol (Macaskie et al., 1995 ) limitation but not in P-, N- or glucose-limited cultures. Biofilm formation is associated with the synthesis of extracellular polymers, which is energetically demanding and carbon-expensive (Chakrabarty, 1996
), and cells usually attempt to conserve available carbon for essential functions (Harder & Dijkhuizen, 1983
), which mainly involves minimizing the diversion of substrate carbon into extracellular polymeric substances (EPS) (Tempest & Wouters, 1981
). It was shown previously that the composition of the EPS of this organism and also the degree of metal-ion-mediated cross-linking are dependent on the growth medium (Bonthrone et al., 2000
). In this study, although negligible biofilm was formed under P and N limitation the initial event in biofilm formation, viz. cellsubstratum attachment (Korber et al., 1995
), did occur. Biofilm continued to accumulate under N-limitation over a longer duration, but the amount was still negligible compared with that obtained under lactose limitation. With limiting glucose, the amount of biofilm was decreased, suggesting that the nature of the limiting carbon source is also important. A glucose-repressive effect may occur, even under prolonged starvation conditions, an observation that would warrant further study.
An electron microscope study of biofilm and planktonic cells grown under the three conditions showed morphological differences. Bacterial cell morphology varies in response to environmental signals, with a reduced cell size commonly promoted as a response to starvation (Mueller, 1996 ). Filamentous Pseudomonas were reported in biofilms (Jensen & Woolfolk, 1985
), possibly promoted by O2-limitation and turbulent conditions. The filamentous cells seen under P-limitation (this study) resemble the elongated Escherichia coli and Salmonella typhimurium swarmer cells described by Harshey & Matsuyuma (1994)
. An increasing number of identified species produce swarmer cells, a phenomenon which also appears to be related to surface colonization (Harshey, 1994
). However, in our study colonization under P-limitation was poor, with only a small number of long cells; furthermore there was no evidence (Fig. 3
) to suggest that microcolony formation was related to long cell occurrence. Electron microscopy showed that the cell surfaces appeared to alter under the different nutrient restrictions, with populations differing in their expression of cell surface appendages, resembling fimbriae (Sharon, 1984
), some of which were designated as a special class (curli), which are implicated in surface colonization and biofilm formation (Olsen et al., 1989
). There is interest in the regulation of fimbrial expression as these structures are also associated with virulence (Curtiss & Kelly, 1987
; Vidal et al., 1998
), their presence facilitating attachment to the host tissues. Regulation of fimbrial expression is strongly affected by environmental conditions, e.g. temperature, external pH, osmolarity and nutrient status (van der Woude et al., 1989
; Schmoll et al., 1990
; Edwards & Schifferli, 1997
; Vanmaele & Armstrong, 1997
; Xie et al., 1997
). In the case of curli expression the subunit gene csgA is regulated by the RpoS sigma factor (Olsen et al., 1993
) and also via the OmpR component of the two-component EnvZ/OmpR sensorregulator, concluded by analysis of adhesive mutants of E. coli selected under continuous culture (Vidal et al., 1998
). Thus, it seems possible that expression of fimbrial adhesins on the cell surface is responsible for the various degrees of biofilm observed here. Accordingly mutants of E. coli and Pseudomonas aeruginosa unable to form biofilm on polyvinyl chloride (PVC) lacked the ability to produce type I and type IV fimbriae, respectively (Pratt & Kolter, 1998
; OToole & Kolter, 1998
), while Salmonella enteritidis defective in biofilm formation on stainless steel and teflon failed to produce thin, aggregative fimbriae (designated SEF 17: Austin et al., 1998
). These studies suggest that more than one fimbrial type is involved in biofilm development (Stickler, 1999
) and, indeed, both the large fimbriae and the curli were absent in the P- or N-limited Citrobacter cells (this study, Fig. 3b
). The observation that fimbrial expression appears to be reduced with glucose as the carbon source, even when cells are C-restricted, suggests a level of control that would warrant future investigation.
Cell surface fimbrial expression does not preclude the possibility that the cell wall composition, or the EPS produced under lactose limitation, may differ in a way that promotes cell-to-cell or cell-to-substratum adhesion. Cell surface composition and chemistry are functions of the cellular environment (Herbert, 1961 ), with many differences reported for cells grown under P and C limitation (Zhan et al., 1991
; Breedveld et al., 1995
; Bonthrone et al., 2000
).
Lactose-limited biofilm shares many features with other biofilms. Wimpenny & Colasanti (1997) reviewed three different conceptual models of biofilm structure: the dense biofilms model, the heterogeneous mosaic model (Keevil & Walker, 1992
) and the water channel model (Costerton et al., 1994
) by which most biofilms may be described. Of these, the last is the most suitable to describe the Citrobacter lactose-limited biofilm, the thickness of which corresponds well with depth measurements reported for other biofilms, e.g. that of a Klebsiella pneumoniae/P. aeruginosa mixed population (Murga et al., 1995
), although the Citrobacter biofilm is thick compared to Pseudomonas biofilms (
30 µm: Stewart et al., 1993
; Murga et al., 1995
) and is more comparable to the biofilm of Klebsiella pneumoniae (100 µm: Murga et al., 1995
).
In contrast to the lactose-limited biofilm the structure of the glucose-limited Citrobacter biofilm at the same age (Fig. 5) resembled the heterogeneous mosaic biofilm model (Keevil & Walker, 1992
); the majority of the surface was colonized by single cells and discrete colonies, with some growing into stacks. The effect of prolonged culture and the extent to which glucose-mediated regulation plays a role were not investigated further.
In order to attempt to explain the nutrient-limitation-dependent variation in biofilm formation it was considered whether the increased biofilm production was related to increased EPS production. Tait et al. (1986) observed that high C:N and C:P ratios in the medium were associated with high levels of polymer production. However, no difference was found between the amount of polymer extracted from planktonic cells in the culture outflow in the three media (extraction and quantitation methods were as described by Bonthrone et al., 2000
). Hence, there is probably no association between the level of EPS production and the extent of biofilm formation by this strain (but note that there was insufficient biofilm for comparison of EPS of attached cells). In contrast, other studies (Bonthrone et al., 2000
) showed that more EPS was produced from lactose-grown than from glycerol-grown cells in batch culture and that the content of cross-linking cations was higher. Also, the phosphate species of the lipopolysaccharide was different in the two types of cell, determined from the 31P NMR spectra attributed to phosphate groups of the lipid A component of the lipopolysaccharide and differentiated from phospholipids by the NMR spectra (Bonthrone et al., 2000
). The same study showed that material extracted from planktonic phosphate-limited cells from a chemostat gave a different 31P NMR spectrum, with only one major phosphate species instead of three as seen under C-limitation. Clearly there can be some differences in the structure of the extracellular LPS and EPS but since N- and P-limited cells gave similar overall yields of EPS and similar biofilms (this study) differences in the EPS structure and composition are unlikely to play a major role.
A major objective of this work was to achieve good biofilm growth commensurate with high phosphatase activity, since this allows the biofilm to accumulate heavy metals (Macaskie et al., 1995 ; Finlay et al., 1999
); some of the phosphatase enzyme is tethered to cell surface materials (Macaskie et al., 2000
). The phosphatase (PhoN) activity of this strain was investigated previously (e.g. Hambling et al., 1987
; Jeong & Macaskie, 1999
; Finlay et al., 1999
) and this study shows that activity was affected significantly by nutrient limitation, with the highest activity observed under lactose limitation; the values reported for lactose-limited planktonic cells in the outflow (Table 2
) are in good agreement with previous studies (Finlay et al., 1999
). Variation in phosphatase activity between planktonic cells in replicate experiments was noted by Hambling et al. (1987)
and these authors reported a similar difference in activity between C- and P-limited cells (75% decrease in the latter). The effect of N limitation has not been previously described. The activity of PhoN in Salmonella is under the control of the phoP/phoQ sensorregulator system (Miller et al., 1989
) and in the present strain is regulated by both C and P limitation (Butler et al., 1991
; discussed in Jeong & Macaskie, 1999
) with a transient repression in batch cultures seen under glucose addition (Butler et al., 1991
). The results presented here support this finding, as substitution of the lactose carbon source by glucose gave lower planktonic cell phosphatase activity (Table 2
).
Microbial activity can be affected by attachment to a surface (Fletcher, 1991 ). Most examples describe enhanced activity but, conversely, in some cases surface attachment reduces cell activity (Bright & Fletcher, 1983
; Gordon et al., 1983
). Here, Citrobacter phosphatase activity was lower in the lactose-limited biofilm cells as compared to planktonic cells although a previous study using glycerol-limited cells gave a similar activity in both populations (P. Clark & L. E. Macaskie, unpublished). Estermann et al. (1954)
reported that phosphatase activity was depressed by the addition of a surface and the present results suggest that this response is modified by the nature of the carbon source. It could be argued that this is an effect of local pH but no pH gradients were found in lactose-limited biofilms using pH microelectrode probes although lactose-pre-grown biofilms developed a pH gradient (pH 7·0 in the bulk fluid; pH 5 at the surface of the substratum) when subsequently grown-on in P- or N-limited media (Allan et al., 1999
). Low pH was associated with decreased phosphatase production in planktonic cells (Jeong & Macaskie, 1999
). Although the regulation of phoN is under the control of the phoP/phoQ sensorregulator system (Miller et al., 1989
) this study does not attempt to address the question of whether the stimulus is nutrient restriction or is an effect of pH.
This study raises a number of important questions regarding biofilm regulation. It shows that the balance and composition of the medium play a role in determining whether a cell population forms a biofilm or not, and extends previous studies suggesting that phosphatase activity is regulated according to the nutrient conditions and/or pH. Both enzyme activity and biofilm formation are influenced by environmental conditions (correlation of biofilm biomass with planktonic cell phosphatase specific activity, r2=0·988), implying that the genes responsible for both functions (and also, possibly, the production of fimbriae) may be organized within the same stimulon (a stimulon refers to all the operons responding together to an environmental stimulus, regardless of how many regulons and modulons may be involved: Neidhardt et al., 1990 ). Various studies suggest that fimbrial expression is associated with biofilm formation; fimbriae are known to be involved in attachment (Low et al., 1996
; Edwards & Schifferli 1997
; Goluszko et al., 1997
) and biofilm development (Austin et al., 1998
; OToole & Kolter, 1998
; Pratt & Kolter, 1998
) and are likely to be involved in the mechanism of biofilm formation in the present case. Previous studies have shown that regulation of fimbrial production is affected by carbon source, nitrogen source (Edwards & Schifferli, 1997
), pH (Low, 1996
) and temperature (Xie et al., 1997
). Edwards & Schifferli (1997)
have proposed a model which describes colonization of the mammalian gut by enteric bacteria in terms of fimbrial expression and the effects of host environment. This model may warrant further study in the context of biofilms of environmental or biotechnological importance, both as carriers of biotechnologically important enzymes and also as a way to present the maximum biocatalytic surface prior to developing mathematical models of the biofilm surface and integration into exisiting bioreactor process models.
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NOTE ADDED IN PROOF |
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Austin, J. W., Sanders, G., Kay, W. W. & Collinson, S. K. (1998). Thin aggregative fimbriae enhance Salmonella enteritidis biofilm. FEMS Microbiol Lett 162, 2295-2301.
de Beer, D., Roe, F. & Lewandowski, Z. (1994). Effects of biofilm structures on oxygen distribution and mass transport. Biotechnol Bioeng 43, 1131-1138.
Bonthrone, K. M., Quarmby, J., Hewitt, C. J., Allan, V. M. J., Paterson-Beedle, M., Kennedy, J. F. & Macaskie, L. E. (2000). The effect of the growth medium on the composition and metal binding behaviour of the extracellular polymeric material of a metal-accumulating Citrobacter sp. Environ Technol 21, 123-134.
Breedveld, M. W., Benesi, A. J., Marco, M. L. & Miller, K. J. (1995). Effect of phosphate limitation on the synthesis of periplasmic cyclic ß-(1,2)-glucans. Appl Environ Microbiol 61, 1045-1053.[Abstract]
Bright, J. J. & Fletcher, M. (1983). Amino acid assimilation and electron transport system activity in attached and free living marine bacteria. Appl Environ Microbiol 45, 818-825.
Bryers, J. D. (1994). Biofilms and the technological implications of microbial cell adhesion. Colloids Surf B Biointerfaces 2, 9-23.
Butler, A. J., Hallett, D. S. & Macaskie, L. E. (1991). Phosphatase production by a Citrobacter sp. growing in batch culture and use of batch cultures to investigate some limitations in the use of polyacrylamide gel-immobilised cells for product release. Enzyme Microb Technol 13, 716-721.
Chakrabarty, A. M. (1996). Why and how P. aeruginosa makes alginate under starvation conditions. In Microbial Biofilms (Proceedings, ASM Conference, Snowbird, Utah, USA). Washington, DC: American Society for Microbiology.
Costerton, J. W., Lewandowski, Z., Caldwell, D. E., Korber, D. R., de Beer, D. & James, G. (1994). Biofilms: the customised microniche. J Bacteriol 176, 2137-2142.[Medline]
Curtiss, R.III & Kelly, S. M. (1987). Salmonella typhimurium deletion mutants lacking adenylate cyclase and cyclic AMP receptor protein are avirulent and immunogenic. Infect Immun 55, 3035-3043.[Medline]
Dalton, H. M., Poulsen, L. K., Halasz, P., Angles, M. L., Goodman, A. E. & Marshall, K. C. (1994). Substrate-induced morphological changes in a marine bacterium and their relevance to biofilm structure. J Bacteriol 176, 6900-6906.[Abstract]
Davies, D. G., Parsek, M. R., Pearson, J. P., Iglewski, B. H., Costerton, J. W. & Greenberg, E. P. (1998). The involvement of cell-to-cell signals in the development of a bacterial biofilm. Science 280, 295-298.
Edwards, R. A. & Schifferli, D. M. (1997). Differential regulation of fasA and fasH expression of Escherichia coli 987P fimbriae by environmental cues. Mol Microbiol 25, 797-809.[Medline]
Ellwood, D. C., Keevil, C. W., Marsh, P. D., Brown, C. M. & Wardell, J. D. (1982). Surface-associated growth. Philos Trans R Soc Lond Ser B Biol Sci 297, 517-523.[Medline]
Estermann, E. F., Peterson, G. H. & McLaren, A. D. (1954). Digestion of clayprotein, ligninprotein and silicaprotein complexes by enzymes and bacteria. Proc Soil Sci Soc Am 23, 31-36.
Finlay, J. A., Allan, V. J. M., Conner, A., Callow, M. E., Basnakova, G. & Macaskie, L. E. (1999). Phosphate release and heavy metal accumulation by biofilm-immobilised and chemically-coupled cells of a Citrobacter sp. pre-grown in continuous culture. Biotechnol Bioeng 63, 87-97.[Medline]
Fletcher, M. (1991). The physiological activity of bacteria attached to solid surfaces. Adv Microb Physiol 32, 53-80.[Medline]
Goluszko, P., Popov, V., Selvarangan, R., Nowicki, S., Pham, T. & Norwicki, B. J. (1997). The Dr fimbriae operon of uropathogenic Escherichia coli mediate microtubule-dependent invasion to the HeLa epithelial line. J Infect Dis 176, 158-67.[Medline]
Gordon, A., Gerchakov, S. M. & Millero, F. J. (1983). Effects of inorganic particles on metabolism by a periphytic marine bacterium. Appl Environ Microbiol 45, 411-417.
Hambling, S. G., Macaskie, L. E. & Dean, A. C. R. (1987). Phosphatase synthesis in a Citrobacter sp. J Gen Microbiol 133, 2743-2749.
Harder, W. & Dijkhuizen, L. (1983). Physiological responses to nutrient limitation. Annu Rev Microbiol 37, 1-23.[Medline]
Harshey, R. M. (1994). Bees arent the only ones: swarming in Gram-negative bacteria. Mol Microbiol 13, 393-394.
Harshey, R. M. & Matsuyama, T. (1994). Dimorphic transition in E. coli and S. typhimurium: surface induced differentiation into hyperflagellate swarmer cells. Proc Natl Acad Sci USA 91, 8631-8635.[Abstract]
Herbert, D. (1961). The chemical composition of micro-organisms as a function of their environment. Symp Soc Exp Biol Med 38, 391-416.
Huang, C., Peretti, S. W. & Bryers, J. D. (1994). Effects of medium carbon-to-nitrogen ratio on biofilm formation and plasmid stability. Biotechnol Bioeng 44, 329-336.
Huang, C. T., Xu, K. D., McFeters, G. A. & Stewart, P. S. (1998). Spatial patterns of alkaline phosphatase expression within bacterial colonies and biofilms in response to phosphate starvation. Appl Environ Microbiol 64, 1526-1531.
James, G. A., Beaudette, L. & Costerton, J. W. (1995). Interspecies bacterial interactions in biofilms. J Ind Microbiol 15, 257-262.
Janning, K. J., Harremoes, P. & Nielson, M. (1995). Evaluating and modelling the kinetics in a full scale submerged denitrification filter. Water Sci Technol 32, 115-13.
Jensen, R. H. & Woolfolk, C. A. (1985). Formation of filaments by Pseudomonas putida. Appl Environ Microbiol 50, 364-372.
Jeong, B. C. & Macaskie, L. E. (1999). Production of two phosphatases by a Citrobacter sp. grown in batch and continuous culture. Enzyme Microb Technol 24, 218-224.
Jeong, B. C., Bonthrone, K. M., Hawes, C. & Macaskie, L. E. (1997). Localization of enzymically enhanced heavy metal accumulation by Citrobacter sp. and metal accumulation in vitro by liposomes containing entrapped enzyme. Microbiology 143, 2497-2507.[Abstract]
Keevil, C. W. & Walker, J. T. (1992). Nomarski DIC microscopy and image analysis of biofilm. Bin Comput Microbiol 4, 93-95.
Korber, D. R., Lawrence, J. R., Lappin-Scott, H. M. & Costerton, J. W. (1995). Growth of microorganisms on surfaces. In Microbial Biofilms , pp. 1-38. Edited by H. Lappin-Scott & J. W. Costerton. Cambridge:Cambridge University Press.
Kugaprasatham, S., Nagaoka, H. & Ohgaki, S. (1992). Effect of turbulence on nitrifying biofilms at non-limiting substrate conditions. Water Res 26, 1629-1638.
Lawrence, J. R., Korber, D. R., Hoyle, B. H., Costerton, J. W. & Caldwell, D. E. (1991). Optical sectioning of microbial biofilms. J Bacteriol 173, 6558-6567.[Medline]
Lewandowski, Z. (1998). Structure and function of bacterial biofilms. Corrosion 295, 1-15.
van Loosdrecht, M. C. M., Eikelboom, D., Gjaltema, A., Mulder, A., Tijhuis, L. & Heijnen, J. J. (1995). Biofilm structures. Water Sci Technol 32, 35-43.
Low, D., Braaten, B. & van der Woude, M. (1996). Fimbriae. In Escherichia coli and Salmonella, pp. 146158. Edited by F. C. Neidhardt and others. Washington, DC: American Society for Microbiology.
Macaskie, L. E., Empson, R. M., Lin, F. & Tolley, M. R. (1995). Enzymically-mediated uranium accumulation and uranium recovery using a Citrobacter sp. immobilised as a biofilm within a plug-flow reactor. J Chem Technol Biotechnol 63, 1-16.
Macaskie, L. E., Bonthrone, K. M., Yong, P. & Goddard, D. (2000). Enzymatically-mediated bioprecipitation of uranium by a Citrobacter sp.: a concerted role for exocellular lipo-polysaccharide and associated phosphatase in biomineral formation. Microbiology 146, 1855-1867.
Miller, S. I., Kukral, A. M. & Mekalanos, J. J. (1989). A two component regulatory system (phoP/phoQ) controls Salmonella typhimurium virulence Proc Natl Acad Sci USA 86, 5054-5058.[Abstract]
Mueller, R. F. (1996). Bacterial transport and colonisation in low nutrient environments. Water Res 30, 2681-2690.
Murga, R., Stewart, P. S. & Daly, D. (1995). Quantitative analysis of biofilm thickness variability. Biotechnol Bioeng 45, 503-510.
Neidhardt, F. C., Ingraham, J. L. & Schaechter, M. (1990). Regulation of gene expression: multigene systems and global regulation. In Physiology of the Bacterial Cell , pp. 351-388. Edited by F. C. Neidhardt. Sunderland, MA:Sinauer Associates.
Ohashi, A., Viraj de Silva, D. J., Mobarry, B., Manem, J. A., Stahl, D. A. & Rittmann, B. E. (1995). Influence of substrate C/N ratio on the structure of multispecies biofilms consisting of nitrifiers and heterotrophs. Water Sci Technol 32, 75-84.
Olsen, A., Jonsson, A. & Normark, S. (1989). Fibronectin binding mediated by a novel class of surface organelles on Escherichia coli. Nature 338, 652-655.[Medline]
Olsen, A., Arnqvist, A., Hammar, M., Sukupolvi, S. & Nomark, S. (1993). The RpoS sigma factor relieves H-NS-mediated transcriptional repression of csgA, the subunit gene of fibronectin-binding curli in Escherichia coli. Mol Microbiol 7, 523-536.[Medline]
OToole, G. A. & Kolter, R. (1998). Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol Microbiol 30, 295-305.[Medline]
Pattanapipitpaisal, P., Mabbett, A. N., Finlay, J. A. & 10 other authors (2001). Reduction of Cr(VI) and bioaccumulation of chromium by Gram positive and Gram negative microorganisms not previously exposed to Cr-stress. Environ Technol (in press).
Peyton, B. M. (1996). Effects of shear stress and substrate loading rate on Pseudomonas aeruginosa biofilm thickness and density. Water Res 30, 29-36.
Pratt, L. A. & Kolter, R. (1998). Genetic analysis of Escherichia coli biofilm formation, roles of flagella, motility, chemotaxis and type I pili. Mol Microbiol 30, 285-293.[Medline]
Schmoll, T., Ott, M., Oudega, B. & Hacker, J. (1990). Use of a wild-type gene fusion to determine the influence of environmental conditions on expression of the S fimbrial adhesin in an Escherichia coli pathogen. J Bacteriol 172, 5103-5111.[Medline]
Sharon, N. (1984). Lectin-like bacterial adherence to animal cells. In Attachment of Organisms to the Gut Mucosa , pp. 129-147. Edited by E. C. Boedeker. Boca Raton, FL:CRC Press.
Stewart, P. S., Peyton, B. M., Drury, W. J. & Murga, R. (1993). Quantitative observations of heterogeneities in Pseudomonas aeruginosa biofilms. Appl Environ Microbiol 59, 327-329.[Abstract]
Stickler, D. (1999). Biofilms. Curr Opin Microbiol 2, 270-275.[Medline]
Tait, M. I., Sutherland, I. W. & Clarkesturman, A. J. (1986). Effect of growth conditions on the production, composition and viscosity of Xanthomonas campestris exopolysaccharide. J Gen Microbiol 132, 1483-1492.
Tempest, D. W. & Wouters, J. T. M. (1981). Properties and performance of microorganisms in chemostat culture. Enzyme Microb Technol 3, 283-290.
Vanmaele, R. P. & Armstrong, G. D. (1997). The effect of carbon source on localised adherence of enteropathogenic Escherichia coli. Infect Immun 65, 1408-1413.[Abstract]
Vidal, O., Longrin, R., Prigent-Combarat, C., Dorel, C., Hooreman, M. & Lejeune, P. (1998). Isolation of an Escherichia coli K-12 mutant strain able to form biofilms on inert surfaces: involvement of a new ompR allele that increases curli expression. J Bacteriol 180, 2442-2449.
Wimpenny, J. W. T. & Colasanti, R. (1997). A unifying hypothesis for the structure of microbial biofilms based on cellular automaton models. FEMS Microbiol Ecol 22, 1-16.
van der Woude, M. W., de Graf, F. K. & van Verseveld, H. W. (1989). Production of the fimbrial adhesin 987P by enterotoxigenic Escherichia coli during growth under controlled conditions in a chemostat. J Gen Microbiol 135, 3421-3429.[Medline]
Xie, H., Cai, S. & Lamont, R. J. (1997). Environmental regulation of fimbrial gene expression in Porphyromonas gingivalis. Infect Immun 65, 2265-2271.[Abstract]
Zhan, H., Chang Lee, C. & Leigh, J. A. (1991). Induction of the second exopolysaccharide (EPSb) in Rhizobium meliloti SU-47 by low phosphate concentrations. J Bacteriol 173, 7391-7394.[Medline]
Received 6 December 2000;
revised 16 August 2001;
accepted 30 August 2001.
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