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Osmotic stimulation of microcolony development by Nitrosomonas europaea
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Osmotic stimulation of microcolony development by Nitrosomonas europaea Nicholas J. Wood, Jan SÖrensen * Section of Genetics and Microbiology, Department of Ecology and Molecular Biology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark Received 29 March 1998; revised 29 June 1998; accepted 29 June 1998
Abstract In liquid cultures low levels of salt (0.1 M NaCl) had no effect on the growth rate of proliferating cells of Nitrosomonas europaea (NCIMB 11850), while at higher salt concentrations the growth rate was progressively reduced. A technique which enabled enumeration of microcolony-forming cells after just a few days incubation on the surface of membrane filters was subsequently used to investigate the effects of medium osmolarity and osmoprotectants on the ability to initiate cell division in single cells from liquid cultures. Typically, only V50% of the cells from early stationary phase cultures in basal medium was able to initiate cell division and form microcolonies during subsequent incubation on filters on the same medium. However, the fraction of cells forming microcolonies was stimulated by low levels of salt, while higher concentrations were inhibitory. A range of bacterial compatible solutes (betaine, trehalose, proline, MOPS, taurine and GABA) were unable to enhance microcolony formation at inhibitory salt concentrations. Other solutes (KCl, LiCl, mannitol, sucrose) also stimulated microcolony development, demonstrating that the effect was osmotic. A low salt (0.1 M NaCl) shock prior to incubation at inhibitory salt levels dramatically increased the number of cells forming microcolonies. A comparative study of several Nitrosospira spp. indicated that osmotic stimulation of microcolony development may be specific to N. europaea, although the proportion of cells able to form microcolonies was much smaller in the Nitrosospira spp. examined. Taken together these results suggest that while inhibition of growth in N. europaea by high osmolarity may be similar to that observed in other bacteria, the osmotic stimulation observed for initiation of cell division may have important implications for the successful establishment and recovery of this nitrifier in biofilms and in soil. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Ammonia-oxidizing bacterium; Nitrosomonas europaea; Microcolony; Surface-attached growth; Osmoregulation ; Compatible solute
1. Introduction The ammonia oxidizers are a group of chemolithotrophic organisms whose members are divided * Corresponding author. Tel.: +45 35282626; Fax: +45 35282606; E-mail: [email protected]
between the L and Q subdivisions of the Proteobacteria [1,2]. Recent molecular studies have suggested a high level of diversity within populations of these organisms in soil, estuarine and marine environments [3^7]. Several halotolerant, halophilic and obligately halophilic species exist [8,9] and the level of salinity is an important regulatory factor in the activity of
0168-6496 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 6 4 9 6 ( 9 8 ) 0 0 0 6 6 - X
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some nitri¢ers, having been shown to both stimulate and inhibit their activity [10^12]. Water potential a¡ects the activity of nitri¢ers in the soil. Stark and Firestone [13] described two effects of water potential on ammonia oxidation: at water potentials greater than 30.6 MPa the major e¡ect was a limitation in the substrate supply due to longer di¡usion paths, while at water potentials lower than 30.6 MPa the most important factor was a detrimental e¡ect on cell activity due to dehydration of the cell cytoplasm. To an extent bacteria can overcome the deleterious e¡ects of water stress by osmoregulation, accumulating compatible solutes which protect intracellular metabolic functions from the adverse e¡ects of high levels of internal solutes. Such compounds are accumulated by synthesis and/ or transport in a wide range of bacteria living in diverse environments [14]. If, when supplied exogenously, they relieve the e¡ects of high osmolarity, they are termed osmoprotectants. In the soil, ammonia-oxidizing bacteria (AOB) are frequently associated with surfaces [15,16] and in sewage with large aggregates of cells [17]. The factors controlling activity and growth on surfaces are poorly de¢ned but their physiology may be very different from that of cells in suspension. Surface-associated nitrifying bacteria are active at lower pH values and more resistant to nitri¢cation inhibitors than liquid cultures of cells [18,19]. However, due to their low growth rates it is di¤cult to culture AOB on solid surfaces, colony formation on agar plates typically taking several months. This has deterred investigations into the e¡ects of physical and chemical phenomena such as osmolarity and osmoprotectants on the process of colony formation on surfaces. In the present study, we employed a membrane ¢lter technique [20] to investigate the e¡ect of osmolarity on initiation of cell division on surfaces (microcolony development) by single cells of a soil isolate of Nitrosomonas europaea and several Nitrosospira species. The method o¡ers several bene¢ts over traditional methods for studies of surface-attached growth of nitri¢ers, such as the ability to move cells rapidly from one set of conditions to another and increase the number of replicates. Growth typically takes only 4^5 days and contamination problems may be more easily overcome. We present evidence of a di¡erence between surface-attached and sus-
pended cells in terms of growth under di¡erent osmotic regimes.
2. Materials and methods 2.1. Bacterial strains and culture conditions A strain of N. europaea (NCIMB 11850) isolated from soil by R.M. Macdonald of Rothamsted Experimental Station, Harpenden, Hertfordshire, UK was employed. Four Nitrosospira species (L115, 40KI, î got Aakra of B6 and AF) [21] were supplied by A î s. All bacthe Agricultural University of Norway, A teria were grown and maintained in basal autotrophic medium (per liter: HEPES, 4 g; (NH4 )2 SO4 , 0.5 g; KH2 PO4 , 0.2 g; CaCl2 W2H2 O, 0.02 g; MgSO4 W7H2 O, 0.04 g; FeNaEDTA, 3.8 mg; phenol red, 1 mg; trace element solution (per 100 ml: NaMoO4 W2H2 O, 10 mg; MnCl2 W2H2 O, 20 mg; CoCl2 W6H2 O, 0.2 mg; ZnSO4 W7H2 O, 10 mg; CuSO4 W5H2 O, 2 mg), 1 ml). Medium pH was adjusted to 7.5 and, after autoclaving, NaHCO3 was added to a ¢nal concentration of 1.5 mM. Maintenance cultures were grown in 50-ml screw cap tubes at 30³C in the dark. Experimental cultures were grown in the same manner using 1% v/v of an early stationary phase inoculum. The osmoprotectants betaine hydrochloride, D(+)trehalose, L-proline, MOPS (3-[N-morpholino]propanesulfonic acid), taurine (2-aminoethanesulfonic acid) and GABA (Q-amino-n-butyric acid), some of which are commonly observed in bacteria [22^26], were added to autoclaved media from concentrated, ¢lter-sterilized stock solutions to a working concentration of 1 mM. All chemicals were purchased from Sigma. 2.2. Measurement of nitrite levels in culture media Nitrite was quanti¢ed to follow growth, using a colorimetric assay in microtiter plates. Culture medium (200 Wl) was added to 40 Wl color reagent (per liter: sulfanilamide, 10 g; N-1-naphthylethylenediamine hydrochloride, 0.5 g; concentrated phosphoric acid, 100 ml). After 15 min incubation at room temperature the absorbance at 540 nm was recorded on a Bio-Kinetics plate reader (Bio-Tek Instruments)
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and compared with a range of standard nitrite solutions. 2.3. Growth of microcolonies on membrane ¢lters Samples of an early stationary phase culture were ¢ltered onto white polycarbonate membrane ¢lters (0.2 Wm pore, 25 mm diameter; Poretics Products, Livermore, CA) before transfer to the surface of 5 ml of medium in multi-well dishes. Standard incubation conditions for microcolony growth on the ¢lters were 5 days at 30³C in the dark. Following incubation, all ¢lters were transferred to the surface of a 0.04% w/v acridine orange solution for staining. After 2 min, ¢lters were washed by two successive transfers to water in Petri dishes (2 min each). After drying (V10 min) the ¢lters were mounted in immersion oil on microscope slides and the number of microcolony-forming units (CFU) in 100 randomly selected ¢elds of view per ¢lter was counted under epi£uorescence illumination on a Zeiss Axioscope microscope. A microcolony was de¢ned as a minimum of four adjacent cells as such clusters were only very rarely seen in suspended cultures. 2.4. Reproducibility and presentation of results Unless otherwise stated results are presented as the mean including standard deviation (S.D.) of three replicate treatments and experiments were performed at least twice. Representative data are shown.
3. Results 3.1. Growth of N. europaea in liquid culture The e¡ect of salt concentration on the growth of N. europaea in liquid culture was investigated using the production of nitrite to follow growth. Low levels of salt had no e¡ect on growth, speci¢c growth rates being 0.072 h31 (S.D. 0.002 h31 ) and 0.075 h (S.D. 0.001 h31 ) in basal medium and basal medium with 0.1 M NaCl respectively (Fig. 1). Higher levels of salt ( s 0.1 M) progressively slowed growth such that in basal medium containing 0.3 M NaCl the speci¢c growth rate fell to 0.046 h31
Fig. 1. In£uence of NaCl concentration (0^0.3 M range) on the speci¢c growth rate of N. europaea in liquid medium. Cells from an early stationary phase culture in basal medium (without NaCl) were used as the inoculum for media containing increasing amounts of salt. The presence (squares) or absence (circles) of an osmoprotectant cocktail containing 1 mM concentrations of betaine hydrochloride, D(+)-trehalose, L-proline, MOPS (3-[N-morpholino]propanesulfonic acid), taurine (2-aminoethanesulfonic acid) and GABA (Q-amino-n-butyric acid) are compared. Data are presented as the standard deviation of the mean of triplicate treatments.
(S.D. 0.001 h31 ) and no growth was observed in cultures containing 0.4 M NaCl. All cultures eventually consumed the whole ammonia pool and produced an equivalent amount of nitrite (data not shown). To determine if exogenously supplied osmoprotectants could relieve the e¡ects of elevated salt levels a panel of compounds encountered in bacteria was added to N. europaea cultures. In basal medium without salt the presence of a cocktail of all the osmoprotectants actually decreased the growth rate of liquid cultures (Fig. 1). As the salt level was increased the inhibitory e¡ect of the cocktail was relieved. When the experiment was repeated over a narrower range of salt concentrations (0^0.1 M in 0.02 M increments) and with the osmoprotectant compounds added separately, it was found that proline was responsible for the growth inhibition at low salt levels. As the osmolarity increased, however, the inhibitory e¡ect was reduced, so that at 0.1 M NaCl the growth rates in basal medium and proline-supplemented medium were comparable (data not shown). At higher salt concentrations ( s 0.1 M) the osmoprotectant cocktail had no e¡ect on growth rates of liquid cultures.
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3.2. Growth of N. europaea as microcolonies on membrane ¢lters Fig. 2 shows the development of N. europaea microcolonies (CFU) on membrane ¢lters on basal medium over a period of 6 days. After 5 days the number of CFU had reached a maximum and no further increase was observed in their number, although cells within microcolonies continued to divide. The exact day on which the number of CFU attained a maximum depended on the batch of cells used but was always within 4^6 days. Even after the number of CFU had reached a maximum, approximately 50% of the original inoculum had failed to form microcolonies. This led to the establishment of standardized incubation conditions of 5 days for the enumeration of nitri¢er cells which could form microcolonies. The e¡ect of salt on microcolony development on membrane ¢lters by N. europaea was di¡erent fro that observed for growth rates in liquid medium. However, it is important to bear in mind when interpreting the results that the two methods used to assess growth (i.e. growth rates in liquid culture and microcolony development on membrane ¢lters) compared two entirely di¡erent criteria. While the former gave an indication of the performance of proliferating cells in a batch culture population, the latter dealt with the ability of any single cell in such a
Fig. 2. Time course of microcolony development of N. europaea on polycarbonate membrane ¢lters. Cells from an early stationary phase liquid culture were transferred to membrane ¢lters which were then £oated on basal medium in multi-well dishes and incubated at 30³C. Filters were subsequently removed, stained for microscopy and the number of microcolonies (CFU) counted on the days shown. Data are presented as the standard deviation of the mean number of microcolonies in 100 randomly selected ¢elds of view from triplicate ¢lters.
Fig. 3. Stimulatory e¡ect of NaCl and other solutes (osmotica) on the development of N. europaea microcolonies (CFU). N. europaea cells from a liquid culture were transferred to membrane ¢lters and then £oated on media which contained di¡erent amounts of NaCl (A) or other solutes that were of equivalent osmocity to 0.1 M NaCl (LiCl and KCl, 0.1 M; mannitol (Man) and sucrose (Suc), 0.18 M) (B). Filters were then incubated under standard conditions (see Section 2). See Fig. 2 for presentation of data.
population to initiate growth and form a microcolony on a membrane surface. Low levels of salt in the culture medium increased the extent of microcolony development while higher ones were inhibitory (Fig. 3A); no microcolony formation was ever observed at the highest salt concentration tested (0.4 M NaCl, data not shown). The level of salt giving rise to the maximum stimulation of microcolony development was always approximately 0.1 M NaCl, which had no e¡ect on the growth rate of liquid cultures. To distinguish between a salt (ionic) e¡ect and an osmotic stress e¡ect in the NaCl-containing medium, we tested other solutes representing osmocities equivalent to 0.1 M NaCl (Fig. 3B). Both ionic (LiCl, KCl) and neutral (mannitol, sucrose) solutes stimulated microcolony development, which clearly indi-
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Fig. 4. Time dependence of NaCl stimulation and inhibition of microcolony formation (CFU) by N. europaea. Cells from a liquid N. europaea culture were transferred to membrane ¢lters which were then incubated at 30³C in the dark on either basal medium (A), 0.1 M NaCl medium (B) or 0.3 M NaCl medium (C). At the times indicated, ¢lters were transferred to either basal medium (dotted lines) or 0.1 M NaCl medium (solid lines) and incubated under standard conditions for 5 days prior to staining. See Fig. 2 for presentation of data.
cated an osmotic e¡ect. However, none of the osmoprotectants tested increased the number of microcolonies which developed at inhibitory salt levels (data not shown). 3.3. Acclimatization to osmotic stress during microcolony growth
Further experiments in which cells on ¢lters were acclimatized by exposure to low salt levels (0.1 M NaCl) prior to incubation at high salt levels (0.25 M NaCl) produced a dramatic increase in the number of microcolonies formed at a normally inhibitory salt concentration (Fig. 5). Again, the time of exposure to the low-salt stimulus appeared to be an im-
To determine if N. europaea could acclimatize to osmotic stress during microcolony development, studies of time-dependent salt exposure and shifts between salt levels were performed. An acclimatization response to osmotic stress could be observed, since low-salt-induced stimulation of microcolony development could still be fully elicited if the cells were transferred back to basal medium without salt after 48 h incubation on medium containing 0.1 M NaCl (Fig. 4, central panel). Similarly, the high-salt inhibition of microcolony development was also found to be dependent on the length of the exposure to 0.3 M NaCl. A rapid decline in microcolony numbers during the ¢rst 4 h of incubation in the presence of 0.3 M NaCl was followed by a slower decline when the exposure time was prolonged (Fig. 4, right hand panel). Recovery of such high-salt-stressed cells as microcolony-forming units was higher when transferred back to 0.1 M NaCl medium than to basal medium.
Fig. 5. Stimulation of N. europaea microcolony growth at inhibitory salt levels by prior incubation on low-salt medium. Cells of N. europaea from a liquid culture were transferred to membrane ¢lters which were then incubated on either 0.1 M NaCl medium (circles and squares) or 0.25 M NaCl medium (triangles). At the times indicated, ¢lters were transferred to either 0.1 M NaCl medium (circles) or 0.25 M NaCl medium (squares and triangles) and incubated at 30³C in the dark for 5 days. Filters were then stained and the number of CFU determined. See Fig. 2 for presentation of results. The experiment was performed once only.
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portant factor in developing maximum culturability at high salt levels, a rapid increase being observed within just 2 h of exposure to 0.1 M NaCl.
4. Discussion
lated from soil. Liquid culture of the strain appeared to accommodate easily modest £uctuations in osmolarity. Hence, increasing the medium salt content from zero to 0.1 M NaCl had little e¡ect on the rate of nitrite production whereas higher amounts of salt progressively reduced the growth rate. While brackish and marine isolates of this species show an obligate salt requirement (optima between 0.3 M and 0.4 M NaCl), strains isolated from other environments such as soil and sewage have no such requirement and optimal concentrations for the culture of these isolates are in the 0^0.1 M NaCl range [8]. The e¡ect of salt on the growth of nitri¢ers thus appears to be at least partially related to the habitat from which they are isolated and our observations on the N. europaea strain are in accordance with the range of optimum salt concentrations for other soil nitri¢er isolates. The explanation for the lack of effect of osmoprotectants at elevated salt levels could of course be that compounds other than those tested are involved for the N. europaea strain. Di¡erent compounds may thus be important in aquatic and terrestrial environments, but a number of those tested (betaine, proline and taurine) have been previously employed in pure culture studies [24,27^29]. Alternatively, N. europaea may simply be incapable of taking up osmoprotectants or maintaining constant turgor during growth at inhibitory osmolarities. Inhibition of growth rate of N. europaea by the osmoprotectant proline at low levels of salt was surprising in the light of a report on the inability of N. europaea to transport this compound [30]. This apparent discrepancy may be a consequence of the af¢nity of the transporter for proline, as we used a 500-fold higher concentration (1 mM as opposed to 2 WM), or it may be a re£ection of di¡erent growth conditions. Hence, the cells used by Frijlink et al. [30] were grown and tested for transport activity at low proline levels, whereas in our experiments the transport system may have been induced when the cells were transferred to growth medium containing high levels of proline.
4.1. The e¡ect of osmolarity on growth rates of liquid cultures
4.2. Low-salt, osmotic stimulation of microcolony development
3.4. Comparative study of microcolony formation by Nitrosospira spp. We ¢nally determined whether the osmotic e¡ect on formation of microcolonies was unique to the N. europaea strain or if it could also be observed in other AOB species. Recent evidence [4,7] has suggested that the Nitrosospira spp. may be numerically important in soils so several strains of this group of ammonia oxidizers were included. Growth of the Nitrosospira strains in liquid culture was very slow when compared to N. europaea, with mean speci¢c growth rates ( þ half-range of duplicate measurements) of 0.015 ( þ 0.001), 0.011 ( þ 0), 0.009 ( þ 0) and 0.009 ( þ 0.001) h31 in basal medium for strains 40KI, L115, AF and B6 respectively. Microscopy was hindered by the tendency of cells of some strains (40KI and B6) to remain joined together following growth in liquid culture, often producing long chains of cells ( s 10^15 Wm). Stimulation of microcolony formation by low salt levels did not appear to occur in Nitrosospira strain L115, which showed only a poor ability to form microcolonies (1 þ 0.6% and 0.4 þ 0.1% of cells forming microcolonies on basal and 0.1 M NaCl medium respectively, mean þ S.D.), although it is di¤cult to be certain of a lack of osmotic e¡ects given that such a low proportion of cells formed microcolonies. Hence, the proportion of cells able to form microcolonies was much lower in Nitrosospira strain L115 than in N. europaea (V1% compared to V50%), even though cells in both cases came from approximately the same stage of growth (early stationary phase). The three other Nitrosospira strains examined (40KI, AF and B6) did not appear to form microcolonies at all within 5 days of growth.
N. europaea (NCIMB 11850) was originally iso-
Whereas growth rates in liquid culture seemed to
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be una¡ected by low salt levels in the 0^0.1 M NaCl range, such low levels of salt clearly stimulated microcolony formation by the N. europaea strain. Using other (non-ionic) solutes it was shown that the stimulation was due to an osmotic e¡ect rather than a salt (ionic) e¡ect of the compounds. It is interesting that the low levels of salt speci¢cally stimulated growth initiation (microcolony development) during surface growth on membrane ¢lters while having no impact on growth rate in liquid culture. The microcolony technique thus provides a unique opportunity to study the stress responses or starvation recovery (initial cell divisions) of single nitri¢er cells. The time course studies of acclimatization to osmotic stress also revealed another interesting aspect of the circumstances that led a cell to form a microcolony. Even after removal of the osmotic stimulus (i.e. low levels of salt) after 48 h incubation, a larger number of microcolonies was formed than in control cultures which were never exposed to salt. This suggests that once a non-dividing cell has received the signal to divide, it becomes committed to that course of development. Remarkably, this signal appears also to function in cells which, following low salt stimulation for just a few hours, are then incubated in the presence of normally inhibitory levels of salt (Fig. 5), leading to a dramatic increase in the level of culturability at high osmolarity. Thus, it appears that within the original inoculum there existed at least two cell types: those that formed microcolonies without any apparent stimulus and those which could be prompted to do so if they experienced an osmotic stimulus. The requirement for additional signals may explain why only 50% of the N. europaea stationary phase cells in a liquid culture were able to initiate growth and develop into microcolonies. Finally, in the comparison with other AOBs, we showed that the osmotic stimulus does not appear to be operational in the Nitrosospira strain L115. Comparison with the other Nitrosospira strains tested was not possible as they failed to form microcolonies within the 5-day incubation period of the experiment. 4.3. High-salt inhibition of microcolony development While the development of microcolonies was dramatically reduced at 0.3 M NaCl compared to basal
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medium, cells in liquid suspension continued to grow, albeit at a much reduced growth rate (0.046 h31 ). Even if it is assumed that the growth rate of nitri¢ers on the ¢lters would have been similarly retarded, the 5-day incubation period would have been su¤cient for a single cell to develop into a microcolony (a minimum of four cells). It appears, therefore, that the inhibition of microcolony formation at high salt concentrations involves other factors and is not only due to an e¡ect on growth rate. Taking the data in Fig. 2 as an example, the growth rate of membrane-attached cells in our experimental system can be approximated from the fact that most cells were well separated from each other at the time of inoculation and that after 24 h of growth most microcolonies consisted of four cells. This gives a speci¢c growth rate of about 0.058 h31 , which is lower than that of suspended cells in basal medium (speci¢c growth rate V0.072 h31 ). As for the stimulation of microcolony formation by low salt levels, the inhibition at high salt levels seemed to involve a direct e¡ect on growth initiation (early cell division) on the ¢lters. In conclusion, the e¡ect of high salt concentrations on both growth rate and microcolony development in N. europaea resembled the general e¡ect of a stress factor, although we did not determine if inhibition was due to salt or osmotic stress. Also the acclimatization studies provided evidence of a general stress response to changes in extracellular salt levels. In many bacteria the response to elevated salt levels is adjustment of the cytoplasmic contents by means of both the synthesis and transport of osmoregulants [14]. Such processes not only increase the growth rate of liquid cultures at inhibitory osmolarities but also restore colony-forming ability on solid growth medium [27,29]. We were unable to show any bene¢t from the external application of a range of osmoprotectants, but this does not exclude the involvement of some form of osmoregulation process. 4.4. Perspectives The work described here is the ¢rst to document both the stimulatory and inhibitory e¡ects of osmolarity on the initiation of growth and subsequent microcolony formation by single cells in axenic cultures of nitrifying bacteria. The microcolony tech-
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nique clearly o¡ers advantages over traditional methods for studying the physiology of surface-attached bacterial populations. For instance, the ability to switch cells rapidly from one set of conditions to another enables the temporal aspects of stimulatory and inhibitory phenomena to be studied more precisely. Our results indicate that the microcolony technique is a rapid, sensitive and convenient means of demonstrating the e¡ects of physical and chemical treatments on the ability of nitrifying bacteria to initiate growth at the single cell level and how changes in such parameters may in£uence the successful establishment of these bacteria in the environment. Soil nitrifying bacteria will be particularly subject to gross £uctuations in water availability and thus salt or osmotic stress, exempli¢ed by periods of drought, freeze-thaw cycles and large, temporary in£uxes of rain or irrigation water. At least one investigation has demonstrated a salt-induced stimulation of surface-attached growth in Nitrosomonas spp. [31]. Changes in the nitri¢er population diversity and activity on the surface of soil particles and plant roots may therefore arise as a result of di¡erential inhibition and stimulation e¡ects on growth by changes in the medium osmolarity.
[5]
[6]
[7]
[8]
[9]
[10]
[11]
Acknowledgments [12]
This work was supported by Grant 9600638 from the Danish Agricultural and Veterinary Science Research Council.
[13]
[14]
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nas europaea colonizing clay minerals from inhibition by nitrapyrin. J. Gen. Microbiol. 137, 1923^1929. Binnerup, S.J., Jensen, D.F., Thordal-Christensen, H. and SÖrensen, J. (1993) Detection of viable, but non-culturable Pseudomonas £uorescens DF57 in soil using a microcolony technique. FEMS Microbiol. Ecol. 12, 97^105. Utaîker, J.B., Bakken, L., Jiang, Q.Q. and Nes, I.F. (1995) Phylogenetic analysis of seven new isolates of ammonia-oxidizing bacteria based on 16S rRNA gene sequences. Syst. Appl. Microbiol. 18, 549^559. Measures, J.C. (1975) Role of amino acids in osmoregulation of non-halophilic bacteria. Nature 257, 398^400. Sutherland, L., Cairney, J., Elmore, M.J., Booth, I.R. and Higgins, C.F. (1986) Osmotic regulation of transcription: induction of the proU betaine transport gene is dependent on accumulation of intracellular potassium. J. Bacteriol. 168, 805^814. McLaggan, D. and Epstein, W. (1991) Escherichia coli accumulates the eukaryotic osmolyte taurine at high osmolarity. FEMS Microbiol. Lett. 81, 209^214. Schleyer, M., Schmid, R. and Bakker, E.P. (1993) Transient, speci¢c and extremely rapid release of osmolytes from grow-
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[31]
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ing cells of Escherichia coli K-12 exposed to hypoosmotic shock. Arch. Microbiol. 160, 424^431. Cayley, S., Record, M.T. and Lewis, B.A. (1989) Accumulation of 3-(N-morpholino)propanesulfonate by osmotically stressed Escherichia coli K-12. J. Bacteriol. 171, 3597^3602. Graham, J.E. and Wilkinson, B.J. (1992) Staphylococcus aureus osmoregulation: roles for choline, glycine betaine, proline and taurine. J. Bacteriol. 174, 2711^2716. Cairney, J., Booth, I.R. and Higgins, C.F. (1985) Salmonella typhimurium proP gene encodes a transport system for the osmoprotectant betaine. J. Bacteriol. 164, 1218^1223. Roth, W.G., Leckie, M.P. and Dietzler, D.N. (1988) Restoration of colony-forming activity on osmotically stressed Escherichia coli by betaine. Appl. Environ. Microbiol. 54, 3142^ 3146. Frijlink, M.J., Abee, T., Laanbroek, H.J., de Boer, W. and Konings, W.N. (1992) Secondary transport of amino acids in Nitrosomonas europaea. Arch. Microbiol. 157, 389^393. Hovanec, T.A. and DeLong, E.F. (1996) Comparative analysis of nitrifying bacteria associated with freshwater and marine aquaria. Appl. Environ. Microbiol. 62, 288^2896.
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Osmotic stimulation of microcolony development by Nitrosomonas europaea Nicholas J. Wood, Jan SÖrensen * Section of Genetics and Microbiology, Department of Ecology and Molecular Biology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark Received 29 March 1998; revised 29 June 1998; accepted 29 June 1998
Abstract In liquid cultures low levels of salt (0.1 M NaCl) had no effect on the growth rate of proliferating cells of Nitrosomonas europaea (NCIMB 11850), while at higher salt concentrations the growth rate was progressively reduced. A technique which enabled enumeration of microcolony-forming cells after just a few days incubation on the surface of membrane filters was subsequently used to investigate the effects of medium osmolarity and osmoprotectants on the ability to initiate cell division in single cells from liquid cultures. Typically, only V50% of the cells from early stationary phase cultures in basal medium was able to initiate cell division and form microcolonies during subsequent incubation on filters on the same medium. However, the fraction of cells forming microcolonies was stimulated by low levels of salt, while higher concentrations were inhibitory. A range of bacterial compatible solutes (betaine, trehalose, proline, MOPS, taurine and GABA) were unable to enhance microcolony formation at inhibitory salt concentrations. Other solutes (KCl, LiCl, mannitol, sucrose) also stimulated microcolony development, demonstrating that the effect was osmotic. A low salt (0.1 M NaCl) shock prior to incubation at inhibitory salt levels dramatically increased the number of cells forming microcolonies. A comparative study of several Nitrosospira spp. indicated that osmotic stimulation of microcolony development may be specific to N. europaea, although the proportion of cells able to form microcolonies was much smaller in the Nitrosospira spp. examined. Taken together these results suggest that while inhibition of growth in N. europaea by high osmolarity may be similar to that observed in other bacteria, the osmotic stimulation observed for initiation of cell division may have important implications for the successful establishment and recovery of this nitrifier in biofilms and in soil. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Ammonia-oxidizing bacterium; Nitrosomonas europaea; Microcolony; Surface-attached growth; Osmoregulation ; Compatible solute
1. Introduction The ammonia oxidizers are a group of chemolithotrophic organisms whose members are divided * Corresponding author. Tel.: +45 35282626; Fax: +45 35282606; E-mail: [email protected]
between the L and Q subdivisions of the Proteobacteria [1,2]. Recent molecular studies have suggested a high level of diversity within populations of these organisms in soil, estuarine and marine environments [3^7]. Several halotolerant, halophilic and obligately halophilic species exist [8,9] and the level of salinity is an important regulatory factor in the activity of
0168-6496 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 6 4 9 6 ( 9 8 ) 0 0 0 6 6 - X
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some nitri¢ers, having been shown to both stimulate and inhibit their activity [10^12]. Water potential a¡ects the activity of nitri¢ers in the soil. Stark and Firestone [13] described two effects of water potential on ammonia oxidation: at water potentials greater than 30.6 MPa the major e¡ect was a limitation in the substrate supply due to longer di¡usion paths, while at water potentials lower than 30.6 MPa the most important factor was a detrimental e¡ect on cell activity due to dehydration of the cell cytoplasm. To an extent bacteria can overcome the deleterious e¡ects of water stress by osmoregulation, accumulating compatible solutes which protect intracellular metabolic functions from the adverse e¡ects of high levels of internal solutes. Such compounds are accumulated by synthesis and/ or transport in a wide range of bacteria living in diverse environments [14]. If, when supplied exogenously, they relieve the e¡ects of high osmolarity, they are termed osmoprotectants. In the soil, ammonia-oxidizing bacteria (AOB) are frequently associated with surfaces [15,16] and in sewage with large aggregates of cells [17]. The factors controlling activity and growth on surfaces are poorly de¢ned but their physiology may be very different from that of cells in suspension. Surface-associated nitrifying bacteria are active at lower pH values and more resistant to nitri¢cation inhibitors than liquid cultures of cells [18,19]. However, due to their low growth rates it is di¤cult to culture AOB on solid surfaces, colony formation on agar plates typically taking several months. This has deterred investigations into the e¡ects of physical and chemical phenomena such as osmolarity and osmoprotectants on the process of colony formation on surfaces. In the present study, we employed a membrane ¢lter technique [20] to investigate the e¡ect of osmolarity on initiation of cell division on surfaces (microcolony development) by single cells of a soil isolate of Nitrosomonas europaea and several Nitrosospira species. The method o¡ers several bene¢ts over traditional methods for studies of surface-attached growth of nitri¢ers, such as the ability to move cells rapidly from one set of conditions to another and increase the number of replicates. Growth typically takes only 4^5 days and contamination problems may be more easily overcome. We present evidence of a di¡erence between surface-attached and sus-
pended cells in terms of growth under di¡erent osmotic regimes.
2. Materials and methods 2.1. Bacterial strains and culture conditions A strain of N. europaea (NCIMB 11850) isolated from soil by R.M. Macdonald of Rothamsted Experimental Station, Harpenden, Hertfordshire, UK was employed. Four Nitrosospira species (L115, 40KI, î got Aakra of B6 and AF) [21] were supplied by A î s. All bacthe Agricultural University of Norway, A teria were grown and maintained in basal autotrophic medium (per liter: HEPES, 4 g; (NH4 )2 SO4 , 0.5 g; KH2 PO4 , 0.2 g; CaCl2 W2H2 O, 0.02 g; MgSO4 W7H2 O, 0.04 g; FeNaEDTA, 3.8 mg; phenol red, 1 mg; trace element solution (per 100 ml: NaMoO4 W2H2 O, 10 mg; MnCl2 W2H2 O, 20 mg; CoCl2 W6H2 O, 0.2 mg; ZnSO4 W7H2 O, 10 mg; CuSO4 W5H2 O, 2 mg), 1 ml). Medium pH was adjusted to 7.5 and, after autoclaving, NaHCO3 was added to a ¢nal concentration of 1.5 mM. Maintenance cultures were grown in 50-ml screw cap tubes at 30³C in the dark. Experimental cultures were grown in the same manner using 1% v/v of an early stationary phase inoculum. The osmoprotectants betaine hydrochloride, D(+)trehalose, L-proline, MOPS (3-[N-morpholino]propanesulfonic acid), taurine (2-aminoethanesulfonic acid) and GABA (Q-amino-n-butyric acid), some of which are commonly observed in bacteria [22^26], were added to autoclaved media from concentrated, ¢lter-sterilized stock solutions to a working concentration of 1 mM. All chemicals were purchased from Sigma. 2.2. Measurement of nitrite levels in culture media Nitrite was quanti¢ed to follow growth, using a colorimetric assay in microtiter plates. Culture medium (200 Wl) was added to 40 Wl color reagent (per liter: sulfanilamide, 10 g; N-1-naphthylethylenediamine hydrochloride, 0.5 g; concentrated phosphoric acid, 100 ml). After 15 min incubation at room temperature the absorbance at 540 nm was recorded on a Bio-Kinetics plate reader (Bio-Tek Instruments)
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and compared with a range of standard nitrite solutions. 2.3. Growth of microcolonies on membrane ¢lters Samples of an early stationary phase culture were ¢ltered onto white polycarbonate membrane ¢lters (0.2 Wm pore, 25 mm diameter; Poretics Products, Livermore, CA) before transfer to the surface of 5 ml of medium in multi-well dishes. Standard incubation conditions for microcolony growth on the ¢lters were 5 days at 30³C in the dark. Following incubation, all ¢lters were transferred to the surface of a 0.04% w/v acridine orange solution for staining. After 2 min, ¢lters were washed by two successive transfers to water in Petri dishes (2 min each). After drying (V10 min) the ¢lters were mounted in immersion oil on microscope slides and the number of microcolony-forming units (CFU) in 100 randomly selected ¢elds of view per ¢lter was counted under epi£uorescence illumination on a Zeiss Axioscope microscope. A microcolony was de¢ned as a minimum of four adjacent cells as such clusters were only very rarely seen in suspended cultures. 2.4. Reproducibility and presentation of results Unless otherwise stated results are presented as the mean including standard deviation (S.D.) of three replicate treatments and experiments were performed at least twice. Representative data are shown.
3. Results 3.1. Growth of N. europaea in liquid culture The e¡ect of salt concentration on the growth of N. europaea in liquid culture was investigated using the production of nitrite to follow growth. Low levels of salt had no e¡ect on growth, speci¢c growth rates being 0.072 h31 (S.D. 0.002 h31 ) and 0.075 h (S.D. 0.001 h31 ) in basal medium and basal medium with 0.1 M NaCl respectively (Fig. 1). Higher levels of salt ( s 0.1 M) progressively slowed growth such that in basal medium containing 0.3 M NaCl the speci¢c growth rate fell to 0.046 h31
Fig. 1. In£uence of NaCl concentration (0^0.3 M range) on the speci¢c growth rate of N. europaea in liquid medium. Cells from an early stationary phase culture in basal medium (without NaCl) were used as the inoculum for media containing increasing amounts of salt. The presence (squares) or absence (circles) of an osmoprotectant cocktail containing 1 mM concentrations of betaine hydrochloride, D(+)-trehalose, L-proline, MOPS (3-[N-morpholino]propanesulfonic acid), taurine (2-aminoethanesulfonic acid) and GABA (Q-amino-n-butyric acid) are compared. Data are presented as the standard deviation of the mean of triplicate treatments.
(S.D. 0.001 h31 ) and no growth was observed in cultures containing 0.4 M NaCl. All cultures eventually consumed the whole ammonia pool and produced an equivalent amount of nitrite (data not shown). To determine if exogenously supplied osmoprotectants could relieve the e¡ects of elevated salt levels a panel of compounds encountered in bacteria was added to N. europaea cultures. In basal medium without salt the presence of a cocktail of all the osmoprotectants actually decreased the growth rate of liquid cultures (Fig. 1). As the salt level was increased the inhibitory e¡ect of the cocktail was relieved. When the experiment was repeated over a narrower range of salt concentrations (0^0.1 M in 0.02 M increments) and with the osmoprotectant compounds added separately, it was found that proline was responsible for the growth inhibition at low salt levels. As the osmolarity increased, however, the inhibitory e¡ect was reduced, so that at 0.1 M NaCl the growth rates in basal medium and proline-supplemented medium were comparable (data not shown). At higher salt concentrations ( s 0.1 M) the osmoprotectant cocktail had no e¡ect on growth rates of liquid cultures.
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3.2. Growth of N. europaea as microcolonies on membrane ¢lters Fig. 2 shows the development of N. europaea microcolonies (CFU) on membrane ¢lters on basal medium over a period of 6 days. After 5 days the number of CFU had reached a maximum and no further increase was observed in their number, although cells within microcolonies continued to divide. The exact day on which the number of CFU attained a maximum depended on the batch of cells used but was always within 4^6 days. Even after the number of CFU had reached a maximum, approximately 50% of the original inoculum had failed to form microcolonies. This led to the establishment of standardized incubation conditions of 5 days for the enumeration of nitri¢er cells which could form microcolonies. The e¡ect of salt on microcolony development on membrane ¢lters by N. europaea was di¡erent fro that observed for growth rates in liquid medium. However, it is important to bear in mind when interpreting the results that the two methods used to assess growth (i.e. growth rates in liquid culture and microcolony development on membrane ¢lters) compared two entirely di¡erent criteria. While the former gave an indication of the performance of proliferating cells in a batch culture population, the latter dealt with the ability of any single cell in such a
Fig. 2. Time course of microcolony development of N. europaea on polycarbonate membrane ¢lters. Cells from an early stationary phase liquid culture were transferred to membrane ¢lters which were then £oated on basal medium in multi-well dishes and incubated at 30³C. Filters were subsequently removed, stained for microscopy and the number of microcolonies (CFU) counted on the days shown. Data are presented as the standard deviation of the mean number of microcolonies in 100 randomly selected ¢elds of view from triplicate ¢lters.
Fig. 3. Stimulatory e¡ect of NaCl and other solutes (osmotica) on the development of N. europaea microcolonies (CFU). N. europaea cells from a liquid culture were transferred to membrane ¢lters and then £oated on media which contained di¡erent amounts of NaCl (A) or other solutes that were of equivalent osmocity to 0.1 M NaCl (LiCl and KCl, 0.1 M; mannitol (Man) and sucrose (Suc), 0.18 M) (B). Filters were then incubated under standard conditions (see Section 2). See Fig. 2 for presentation of data.
population to initiate growth and form a microcolony on a membrane surface. Low levels of salt in the culture medium increased the extent of microcolony development while higher ones were inhibitory (Fig. 3A); no microcolony formation was ever observed at the highest salt concentration tested (0.4 M NaCl, data not shown). The level of salt giving rise to the maximum stimulation of microcolony development was always approximately 0.1 M NaCl, which had no e¡ect on the growth rate of liquid cultures. To distinguish between a salt (ionic) e¡ect and an osmotic stress e¡ect in the NaCl-containing medium, we tested other solutes representing osmocities equivalent to 0.1 M NaCl (Fig. 3B). Both ionic (LiCl, KCl) and neutral (mannitol, sucrose) solutes stimulated microcolony development, which clearly indi-
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Fig. 4. Time dependence of NaCl stimulation and inhibition of microcolony formation (CFU) by N. europaea. Cells from a liquid N. europaea culture were transferred to membrane ¢lters which were then incubated at 30³C in the dark on either basal medium (A), 0.1 M NaCl medium (B) or 0.3 M NaCl medium (C). At the times indicated, ¢lters were transferred to either basal medium (dotted lines) or 0.1 M NaCl medium (solid lines) and incubated under standard conditions for 5 days prior to staining. See Fig. 2 for presentation of data.
cated an osmotic e¡ect. However, none of the osmoprotectants tested increased the number of microcolonies which developed at inhibitory salt levels (data not shown). 3.3. Acclimatization to osmotic stress during microcolony growth
Further experiments in which cells on ¢lters were acclimatized by exposure to low salt levels (0.1 M NaCl) prior to incubation at high salt levels (0.25 M NaCl) produced a dramatic increase in the number of microcolonies formed at a normally inhibitory salt concentration (Fig. 5). Again, the time of exposure to the low-salt stimulus appeared to be an im-
To determine if N. europaea could acclimatize to osmotic stress during microcolony development, studies of time-dependent salt exposure and shifts between salt levels were performed. An acclimatization response to osmotic stress could be observed, since low-salt-induced stimulation of microcolony development could still be fully elicited if the cells were transferred back to basal medium without salt after 48 h incubation on medium containing 0.1 M NaCl (Fig. 4, central panel). Similarly, the high-salt inhibition of microcolony development was also found to be dependent on the length of the exposure to 0.3 M NaCl. A rapid decline in microcolony numbers during the ¢rst 4 h of incubation in the presence of 0.3 M NaCl was followed by a slower decline when the exposure time was prolonged (Fig. 4, right hand panel). Recovery of such high-salt-stressed cells as microcolony-forming units was higher when transferred back to 0.1 M NaCl medium than to basal medium.
Fig. 5. Stimulation of N. europaea microcolony growth at inhibitory salt levels by prior incubation on low-salt medium. Cells of N. europaea from a liquid culture were transferred to membrane ¢lters which were then incubated on either 0.1 M NaCl medium (circles and squares) or 0.25 M NaCl medium (triangles). At the times indicated, ¢lters were transferred to either 0.1 M NaCl medium (circles) or 0.25 M NaCl medium (squares and triangles) and incubated at 30³C in the dark for 5 days. Filters were then stained and the number of CFU determined. See Fig. 2 for presentation of results. The experiment was performed once only.
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portant factor in developing maximum culturability at high salt levels, a rapid increase being observed within just 2 h of exposure to 0.1 M NaCl.
4. Discussion
lated from soil. Liquid culture of the strain appeared to accommodate easily modest £uctuations in osmolarity. Hence, increasing the medium salt content from zero to 0.1 M NaCl had little e¡ect on the rate of nitrite production whereas higher amounts of salt progressively reduced the growth rate. While brackish and marine isolates of this species show an obligate salt requirement (optima between 0.3 M and 0.4 M NaCl), strains isolated from other environments such as soil and sewage have no such requirement and optimal concentrations for the culture of these isolates are in the 0^0.1 M NaCl range [8]. The e¡ect of salt on the growth of nitri¢ers thus appears to be at least partially related to the habitat from which they are isolated and our observations on the N. europaea strain are in accordance with the range of optimum salt concentrations for other soil nitri¢er isolates. The explanation for the lack of effect of osmoprotectants at elevated salt levels could of course be that compounds other than those tested are involved for the N. europaea strain. Di¡erent compounds may thus be important in aquatic and terrestrial environments, but a number of those tested (betaine, proline and taurine) have been previously employed in pure culture studies [24,27^29]. Alternatively, N. europaea may simply be incapable of taking up osmoprotectants or maintaining constant turgor during growth at inhibitory osmolarities. Inhibition of growth rate of N. europaea by the osmoprotectant proline at low levels of salt was surprising in the light of a report on the inability of N. europaea to transport this compound [30]. This apparent discrepancy may be a consequence of the af¢nity of the transporter for proline, as we used a 500-fold higher concentration (1 mM as opposed to 2 WM), or it may be a re£ection of di¡erent growth conditions. Hence, the cells used by Frijlink et al. [30] were grown and tested for transport activity at low proline levels, whereas in our experiments the transport system may have been induced when the cells were transferred to growth medium containing high levels of proline.
4.1. The e¡ect of osmolarity on growth rates of liquid cultures
4.2. Low-salt, osmotic stimulation of microcolony development
3.4. Comparative study of microcolony formation by Nitrosospira spp. We ¢nally determined whether the osmotic e¡ect on formation of microcolonies was unique to the N. europaea strain or if it could also be observed in other AOB species. Recent evidence [4,7] has suggested that the Nitrosospira spp. may be numerically important in soils so several strains of this group of ammonia oxidizers were included. Growth of the Nitrosospira strains in liquid culture was very slow when compared to N. europaea, with mean speci¢c growth rates ( þ half-range of duplicate measurements) of 0.015 ( þ 0.001), 0.011 ( þ 0), 0.009 ( þ 0) and 0.009 ( þ 0.001) h31 in basal medium for strains 40KI, L115, AF and B6 respectively. Microscopy was hindered by the tendency of cells of some strains (40KI and B6) to remain joined together following growth in liquid culture, often producing long chains of cells ( s 10^15 Wm). Stimulation of microcolony formation by low salt levels did not appear to occur in Nitrosospira strain L115, which showed only a poor ability to form microcolonies (1 þ 0.6% and 0.4 þ 0.1% of cells forming microcolonies on basal and 0.1 M NaCl medium respectively, mean þ S.D.), although it is di¤cult to be certain of a lack of osmotic e¡ects given that such a low proportion of cells formed microcolonies. Hence, the proportion of cells able to form microcolonies was much lower in Nitrosospira strain L115 than in N. europaea (V1% compared to V50%), even though cells in both cases came from approximately the same stage of growth (early stationary phase). The three other Nitrosospira strains examined (40KI, AF and B6) did not appear to form microcolonies at all within 5 days of growth.
N. europaea (NCIMB 11850) was originally iso-
Whereas growth rates in liquid culture seemed to
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be una¡ected by low salt levels in the 0^0.1 M NaCl range, such low levels of salt clearly stimulated microcolony formation by the N. europaea strain. Using other (non-ionic) solutes it was shown that the stimulation was due to an osmotic e¡ect rather than a salt (ionic) e¡ect of the compounds. It is interesting that the low levels of salt speci¢cally stimulated growth initiation (microcolony development) during surface growth on membrane ¢lters while having no impact on growth rate in liquid culture. The microcolony technique thus provides a unique opportunity to study the stress responses or starvation recovery (initial cell divisions) of single nitri¢er cells. The time course studies of acclimatization to osmotic stress also revealed another interesting aspect of the circumstances that led a cell to form a microcolony. Even after removal of the osmotic stimulus (i.e. low levels of salt) after 48 h incubation, a larger number of microcolonies was formed than in control cultures which were never exposed to salt. This suggests that once a non-dividing cell has received the signal to divide, it becomes committed to that course of development. Remarkably, this signal appears also to function in cells which, following low salt stimulation for just a few hours, are then incubated in the presence of normally inhibitory levels of salt (Fig. 5), leading to a dramatic increase in the level of culturability at high osmolarity. Thus, it appears that within the original inoculum there existed at least two cell types: those that formed microcolonies without any apparent stimulus and those which could be prompted to do so if they experienced an osmotic stimulus. The requirement for additional signals may explain why only 50% of the N. europaea stationary phase cells in a liquid culture were able to initiate growth and develop into microcolonies. Finally, in the comparison with other AOBs, we showed that the osmotic stimulus does not appear to be operational in the Nitrosospira strain L115. Comparison with the other Nitrosospira strains tested was not possible as they failed to form microcolonies within the 5-day incubation period of the experiment. 4.3. High-salt inhibition of microcolony development While the development of microcolonies was dramatically reduced at 0.3 M NaCl compared to basal
181
medium, cells in liquid suspension continued to grow, albeit at a much reduced growth rate (0.046 h31 ). Even if it is assumed that the growth rate of nitri¢ers on the ¢lters would have been similarly retarded, the 5-day incubation period would have been su¤cient for a single cell to develop into a microcolony (a minimum of four cells). It appears, therefore, that the inhibition of microcolony formation at high salt concentrations involves other factors and is not only due to an e¡ect on growth rate. Taking the data in Fig. 2 as an example, the growth rate of membrane-attached cells in our experimental system can be approximated from the fact that most cells were well separated from each other at the time of inoculation and that after 24 h of growth most microcolonies consisted of four cells. This gives a speci¢c growth rate of about 0.058 h31 , which is lower than that of suspended cells in basal medium (speci¢c growth rate V0.072 h31 ). As for the stimulation of microcolony formation by low salt levels, the inhibition at high salt levels seemed to involve a direct e¡ect on growth initiation (early cell division) on the ¢lters. In conclusion, the e¡ect of high salt concentrations on both growth rate and microcolony development in N. europaea resembled the general e¡ect of a stress factor, although we did not determine if inhibition was due to salt or osmotic stress. Also the acclimatization studies provided evidence of a general stress response to changes in extracellular salt levels. In many bacteria the response to elevated salt levels is adjustment of the cytoplasmic contents by means of both the synthesis and transport of osmoregulants [14]. Such processes not only increase the growth rate of liquid cultures at inhibitory osmolarities but also restore colony-forming ability on solid growth medium [27,29]. We were unable to show any bene¢t from the external application of a range of osmoprotectants, but this does not exclude the involvement of some form of osmoregulation process. 4.4. Perspectives The work described here is the ¢rst to document both the stimulatory and inhibitory e¡ects of osmolarity on the initiation of growth and subsequent microcolony formation by single cells in axenic cultures of nitrifying bacteria. The microcolony tech-
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nique clearly o¡ers advantages over traditional methods for studying the physiology of surface-attached bacterial populations. For instance, the ability to switch cells rapidly from one set of conditions to another enables the temporal aspects of stimulatory and inhibitory phenomena to be studied more precisely. Our results indicate that the microcolony technique is a rapid, sensitive and convenient means of demonstrating the e¡ects of physical and chemical treatments on the ability of nitrifying bacteria to initiate growth at the single cell level and how changes in such parameters may in£uence the successful establishment of these bacteria in the environment. Soil nitrifying bacteria will be particularly subject to gross £uctuations in water availability and thus salt or osmotic stress, exempli¢ed by periods of drought, freeze-thaw cycles and large, temporary in£uxes of rain or irrigation water. At least one investigation has demonstrated a salt-induced stimulation of surface-attached growth in Nitrosomonas spp. [31]. Changes in the nitri¢er population diversity and activity on the surface of soil particles and plant roots may therefore arise as a result of di¡erential inhibition and stimulation e¡ects on growth by changes in the medium osmolarity.
[5]
[6]
[7]
[8]
[9]
[10]
[11]
Acknowledgments [12]
This work was supported by Grant 9600638 from the Danish Agricultural and Veterinary Science Research Council.
[13]
[14]
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