Catalase and superoxide dismutase activity in ammonia-oxidising bacteria

Abstract

A range of ammonia-oxidising bacteria (AOB) was tested for the presence of the oxidative stress enzymes catalase and superoxide dismutase (SOD). All AOB possessed both activities, but overall activity and regulation were in several ways different among the strains. Nitrosomonas europaea contained very high specific activities of catalase compared to the other nitrifiers. Only one catalase isozyme, expressed at a constant specific activity per cell during growth in batch culture, was detectable in N. europaea by native PAGE analysis. In contrast, Nitrosospira multiformis possessed four catalase isozymes whose expression was strongly regulated by growth phase in batch culture. SOD expression patterns in the strains generally followed those of catalase, although changes of the SOD isozyme profiles were not apparent in batch culture. Growth of N. europaea on the surface of membrane filters (microcolony and biofilm formation) resulted in altered proportions of catalase and SOD specific activities, indicating regulation by cell density. This report represents the first description of different isozyme composition and of growth phase-dependent regulation of catalase and SOD enzyme production in AOB.

Ammonia-oxidizing bacterium, Catalase, Superoxide dismutase, Oxidative stress, Biofilm

1 Introduction

Despite living in environments which frequently undergo changes in water potential, nutrient availability, temperature and dissolved oxygen, little is known about the response of ammonia-oxidising bacteria (AOB) to a variety of stress factors. This is surprising since AOB are particularly susceptible to inhibition by environmental stress conditions such as a wide range of harmful compounds, light, pH shifts and temperature shifts [1]. Detailed studies of general (global) stress responses and their regulation in AOB have been sparse, although the role in Gram-negative bacteria of heat shock proteins in modifying protein under stress has also been proposed in AOB. Hence, gene sequencing and expression/mutagenesis studies related to the heat shock protein DnaK in Nitrosomonas europaea have recently been reported [2].

As for most environmental bacteria, AOB can be expected to experience oxidative stress from a variety of sources. Specific enzymes, coping directly with harmful compounds such as reactive oxygen species (ROS), may thus be part of a sublethal stress response in AOB. In soil, some organisms produce ROS during antagonistic interactions, but the compounds are also by-products of normal cellular metabolism during aerobic growth where superoxide radicals are generated by NADH dehydrogenase and a number of other cellular systems. Further, hydrogen peroxide may be generated photochemically from organic solutes such as humic acid and other phenolic compounds while Draper and Crosby [3] observed hydrogen peroxide in agricultural runoff waters. These findings suggest that organisms will experience oxidative stress at some point in time and those that are equipped with the best defensive mechanisms will, in an ecological sense, be more successful.

Given that AOB are likely to encounter ROS in their environment, we undertook an investigation of the catalase and superoxide dismutase (SOD) enzyme systems, which have been adopted to cope with ROS in these bacteria. Substantial differences in both the specific activities of enzymes and their regulation between the different AOB were observed. The results present the first demonstration of oxidative stress enzymes, including different patterns of regulation, in AOB.

2 Materials and methods

2.1 Bacterial strains and culture conditions

N. europaea (NCIMB 11850) was supplied by R.M. Macdonald, Rothamsted Experimental Station. Nitrosospira multiformis (NCIMB11849) and Nitrosospira AV (Apple Valley) were supplied by E. Schmidt, University of Minnesota. Ammonia oxidisers were cultured in 500-ml screw cap bottles containing 200 ml medium or 50-ml screw cap tubes containing 20 ml medium and nitrite production was measured to follow growth, all as described previously [4]. Escherichia coli K12 (DSM 498) and Pseudomonas aeruginosa (DSM 50071) were grown aerobically in LB medium at 37°C. Pseudomonas fluorescens DR54 [5] was grown in the same medium but incubated at 30°C. In some experiments oxidative stress was applied to early stationary phase cells by addition of 40 μM hydrogen peroxide (final concentration), which were then incubated for 4 h before harvesting for extraction of enzymes.

In some experiments, AOB cells were grown on white polycarbonate membrane filters (0.2 μm pore, 25 mm diameter; Poretics Products, Livermore, CA, USA) placed on the surface of growth medium. Here, inoculum from an early stationary phase culture was applied at a density of approximately one cell per 100 μm² of filter surface. Filters were then floated on 5 ml of the appropriate medium for 5 days at 30°C in the dark.

2.2 Cell harvest and lysis procedures

Cells (20 ml or 200 ml) were harvested from liquid batch culture by centrifugation and concentrated 10-fold by resuspension in ice-cold potassium phosphate buffer (50 mM phosphate, pH 7.8). After re-centrifugation the pellets were stored at −20°C. Prior to cell lysis, frozen pellets were resuspended in 50 μl phosphate buffer and then subjected to five rounds of sonication in an ice water bath for 20 s followed by cooling for another 20 s. After removal of cellular debris by centrifugation at 4°C the lysate was ready for use.

Filter-grown cells were washed once by flotation of the filters on the surface of 5 ml ice-cold potassium phosphate buffer with gentle shaking for 5 min and the filters were then frozen at −20°C until required. When lysis was performed, filters (usually six) were cut into approximately 5-mm square pieces with scissors and ‘resuspended’ in 400 μl phosphate buffer. Following sonication as above the filter fragments were removed and the lysate was concentrated by centrifugation at 4°C in a Microcon 10 concentrator (10 kDa MW cutoff; Millipore) resulting in a final volume of approximately 50 μl.

Total protein concentration in cell lysates was determined by a modification of the Lowry procedure (Sigma Kit P-5656) using bovine serum albumin as the standard.

2.3 Spectrophotometric catalase and SOD assays

The catalase and SOD specific activities of cell lysates were determined using the spectrophotometric protocols given in [6]. One unit of catalase was defined as the amount of lysate that catalysed the decomposition of 1 μmol H₂O₂ per min at 25°C. One unit of SOD inhibited the rate of xanthine/xanthine oxidase-dependent cytochrome c reduction at 25°C by 50%.

2.4 Native polyacrylamide gel electrophoresis of cell proteins and gel activity staining

Cell lysate (equivalent to one unit of either catalase or SOD activity) was loaded onto a 6% native polyacrylamide Tris-glycine gel (Novex, San Diego, CA, USA) bathed in 1×Tris-glycine buffer, pH 7.0 and the proteins separated at constant voltage (125 V, anodic migration) and room temperature. Subsequently, gels were excised from the cassette, washed for a total of 30 min in 200 ml water (replenished every 5 min) and then stained for either catalase or SOD activity using the protocols described in [7] and [8] respectively. Catalase and SOD activities resulted in achromatic zones on the otherwise uniformly pigmented gel.

3 Results

3.1 Catalase and SOD enzymes in AOB strains

In order to obtain sufficient cell mass, extracts were prepared initially from cells harvested at early stationary phase in liquid batch culture. To compare the results from nitrifier cells with more commonly described organisms, extracts were also prepared from cells of Ps. aeruginosa, Ps. fluorescens and E. coli. As shown in Table 1, all the AOB tested possessed catalase activity. N. europaea possessed comparatively high specific activities of catalase; the activity was approximately 25-fold higher than in the other AOB, Nsp. multiformis and Nitrosospira AV (Table 1). Only Ps. aeruginosa had catalase activities that were as high as those of N. europaea. The Ps. fluorescens strain possessed intermediate activity while E. coli had specific activities that were similar to Nsp. multiformis and Nitrosospira AV. Table 1 also shows that all AOB possessed SOD activity. Specific activities of N. europaea SOD were similar to those of the other nitrifiers, with Nitrosospira AV possessing the highest SOD activity. Ps. aeruginosa had the highest SOD activity of all the organisms tested, while activities in Ps. fluorescens, E. coli and AOB were in the same range.

1

Oxidative stress enzymes in ammonia-oxidising bacteria and some other organisms

Organism	Enzyme activity (units (mg protein)−1)
Catalase	SOD
N. europaeaa	1535±75	71±3
N. europaeab	2504±346	22±6
Nsp. multiformisa	66±9	88±8
Nitrosospira Apple Valleya	62±14	130±9
Ps. aeruginosaa	1335±283	193±37
Ps. fluorescensa	253±38	67±7
E. colia	60±12	42±1

Results are presented as the standard error of the mean of at least triplicate samples.

a

^a Liquid-grown cells from 20-ml batch cultures in 50-ml screw cap tubes, harvested in early stationary phase.

b

^b Surface-grown cells from membrane filters, harvested after biofilm formation.

The isozyme profiles from the cultures were determined after gel chromatographic separation under native conditions and staining for the presence of catalase activity. N. europaea and Nitrosospira AV appeared to contain only a single catalase (Fig. 1A). In contrast, Nsp. multiformis had two isozymes and a further two bands were observed in aged cultures (see below). Gels displayed four zones of SOD activity for N. europaea, although the position of the band with the highest apparent molecular mass coincided with that of a red-brown band, which was possibly a cytochrome (Fig. 1B). Nsp. multiformis lysates had one major SOD band and a faint, minor band whereas Nitrosospira AV had two equally intense bands. Ps. aeruginosa, Ps. fluorescens and E. coli all possessed only one zone of SOD activity under the conditions employed (Fig. 1B).

1

Catalase and SOD isozyme profiles of ammonia-oxidising bacteria. After growth to early stationary phase (20 ml culture volume) the cells were harvested and lysed. Total cell proteins (equivalent to one unit of the appropriate enzyme activity) were separated by native PAGE followed by staining for the presence of either catalase (A) or superoxide dismutase (B) (achromatic zones). The cell extracts present in the lanes are as follows: lane 1, N. europaea; lane 2, Nsp. multiformis; lane 3, Nitrosospira AV; lane 4, E. coli; lane 5, Ps. aeruginosa; lane 6, Ps. fluorescens. The figure is a composite image of several gels but the relative migration rates of the isozymes are comparable.

3.2 Regulation of catalase and SOD enzyme production – effect of growth cycle in AOB batch cultures

The above comparison was based on cells harvested in early stationary phase. To determine whether specific activities and isozyme banding patterns varied with growth phase, cell extracts were prepared from N. europaea and Nsp. multiformis cultures harvested at different points throughout the growth cycle. The regulation of catalase and SOD production differed in the two organisms (Fig. 2). N. europaea produced catalase and SOD at only slightly (max. 2-fold) lower specific activities in exponential phase than in stationary phase during the growth cycle (Fig. 2A). In contrast, when stationary phase cells of Nsp. multiformis (Fig. 2B) were inoculated into fresh growth medium, the catalase specific activities rapidly fell from a high specific activity in the inoculum to a low value of approximately 10–20 units mg⁻¹ protein in the exponentially growing cells. The low catalase level remained until transition phase (progression into stationary phase) when production increased to a value approximately 30-fold over the minimum recorded in exponential phase. Nsp. multiformis also seemed to possess a growth phase-dependent regulation of SOD production, as documented by the higher specific activities after the culture had been growth-arrested for some time. However, the increase in SOD specific activity was less pronounced than that of catalase in stationary phase; the SOD specific activities increased approximately 4-fold when comparing low and high points during the whole growth cycle.

2

Enzyme production during a growth cycle of liquid-grown ammonia-oxidising bacteria. Stationary phase cells of (A) N. europaea and (B) Nsp. multiformis were inoculated into 200 ml growth medium in 500-ml screw cap bottles and nitrite production was followed to measure growth of the cultures (◼). At various times the cultures were sacrificed and cells harvested for later estimation of catalase (●) and SOD (▲) activity. Error bars represent standard errors of the mean from at least three replicate cultures. Lanes 1–5 show the catalase isozyme profiles of N. europaea (C) and Nsp. multiformis (D) harvested after 0, 95, 125, 200 and 410 h, respectively. In panel D the bands A–D represent different catalase isozymes in Nsp. multiformis, as discussed in the text.

The constant catalase specific activity in N. europaea corresponded well with the observation of only a single catalase isozyme throughout the growth cycle (Fig. 2C). Similarly, the constant SOD specific activity in N. europaea was in accordance with an unaltered distribution of the SOD isozyme pattern with four separate bands as referred to above (data not shown). Interestingly, Nsp. multiformis retained the two catalase isozyme bands referred to above during the whole growth cycle (Fig. 2D, bands a and b), but also produced two additional isozyme bands when enzyme production increased dramatically (30-fold) in stationary phase (Fig. 2D, bands c and d). In contrast, no detectable changes occurred to the SOD isozyme pattern during growth of the batch culture (data not shown), despite the 4-fold increase of SOD activity during stationary phase.

3.3 Effect of colony growth on regulation of catalase and SOD enzyme production

Previously, it has been observed that growth on surfaces may affect AOB tolerance to stress (osmotic pressure) [4] so in the present study the effect of surface growth on the production of catalase and SOD isozymes was investigated. The enzymes from N. europaea cells grown for 5 days on the surface of membrane filters were extracted and compared to those of liquid-grown cultures. During incubation the inoculated cells formed microcolonies, and after 5 days a nearly confluent layer of AOB cells formed a thin biofilm on the filters. As shown in Table 1, surface-grown cells produced 2-fold higher and 5-fold lower specific activities of catalase and SOD respectively. However, PAGE analysis of extracts of filter-grown cells, followed by staining for the catalase and SOD isozyme profiles, generated banding patterns similar to those of liquid-grown cells in batch cultures (data not shown). Hence, in N. europaea neither growth phase (liquid cultures) nor surface growth (biofilm cultures) seemed to regulate isozyme expression patterns. Unfortunately, it was not possible to culture Nsp. multiformis to an adequate cell density on the membrane surfaces.

3.4 Effects of reactive oxygen species (H₂O₂) on catalase and SOD specific activities

As suggested above, the changes of enzyme specific activities in liquid-grown and surface-grown cells could be due to different expression levels or direct effects on the enzymes formed. To test the effect of reactive oxygen species (exogenously supplied H₂O₂) on the catalase and SOD specific activities and isozyme patterns, cells of N. europaea and Nsp. multiformis were grown to late exponential phase and then supplemented with 40 μM H₂O₂ for 4 h. After H₂O₂ exposure, no significant differences in either catalase or SOD specific activities were found (data not shown). In addition, H₂O₂ treatment elicited no change to isozyme banding patterns as determined by PAGE (data not shown).

4 Discussion

4.1 Occurrence of oxidative stress enzymes in AOB

Ammonia oxidisers are found in diverse environments such as sewage treatment plants and agricultural soils, undergoing large variations in oxygen status. The formation of ROS has been demonstrated in environments with fluctuating O₂ levels [3], suggesting that bacteria must be capable of detoxifying ROS. Possession of catalase and SOD enzymes in a number of Pseudomonas spp. and other Gram-negative bacteria is well known and the regulation and environmental role of these enzymes have in many cases been studied (e.g. [6,9,10]). In the present study, it was demonstrated that all the AOB studied possessed both catalase and SOD activity, but in very different proportions between strains (Table 1). When cells were harvested in early stationary phase catalase was the major constituent in N. europaea, whilst the other AOB expressed comparable amounts of catalase and SOD per cell. Amongst the other test bacteria, only Ps. aeruginosa produced catalase at a specific activity that was comparable to that in N. europaea.

Only one N. europaea catalase was apparent in the native gels (Fig. 1A) but the N. europaea genome-sequencing project [11] has to date uncovered three sequences with similarity to known catalases. The first of these (gene 97 on contig 472) bears high similarity to the catalase of Desulfovibrio vulgaris. Two further sequences (designated genes 74 and 75 on contig 469) show high similarity to the catalase of the γ-proteobacterium Legionella pneumophila. Although the latter two sequences are currently annotated as two distinct genes, a closer analysis indicates that they are probably a single gene that has either undergone a mutation leading to the introduction of a stop codon or there exists an error in the DNA sequence. If the former were true this would certainly explain the observations in the present work regarding the inability of N. europaea to synthesise anything but a single catalase under any of the environmental conditions tested. Strangely, although four SOD isozymes were observed in native gels, only one SOD gene, highly similar to the iron-containing SOD from Synechocystis sp. (strain PCC6803), has been found by gene sequencing in N. europaea. Cytochromes could interfere with our assay procedure, resulting in false positive bands on gels during the chromogenic assay. Alternatively, this apparent discrepancy between SOD gene sequencing and isozyme gel patterns could be due to the incomplete nature of the genome project. The possibility also exists that ammonia oxidisers may possess unique genes that have not yet been characterised in other organisms. Furthermore, some SOD isozymes may have been formed by post-translational modification.

4.2 Regulation of enzyme synthesis during liquid batch culture

Regulation of the catalase and SOD enzyme synthesis varied depending upon the AOB strain in question. N. europaea apparently possessed a single catalase, which was produced constitutively, i.e. independent of growth phase in the batch cultures. In contrast, catalase synthesis was strongly growth phase-dependent in Nsp. multiformis: catalase activity increased dramatically as the cultures went from rapid growth in exponential phase to slower growth in early stationary phase. The catalase activity attained in stationary phase cells eventually reached the high level found in the inoculum, which was also harvested from stationary phase. Four catalase isozymes were observed in aged stationary phase cultures of the Nsp. multiformis strain, but cells harvested in early stationary phase (200 h, Fig. 2B,D) did not contain isozyme c, which was only found in aged cultures (410 h, Fig. 2B,D). This implies that regulation may occur even in late stationary phase as the culture ages. The possibility obviously arises that the relatively small isozyme c might be a breakdown product of isozyme b, especially when the fall in relative intensity of this band at 410 h is noted. In contrast, isozyme d also appeared in stationary phase but could not be a breakdown product since its apparent molecular mass is higher than any of the other isozymes. This isozyme may therefore be truly regulated by specific induction when the cultures enter stationary phase. By comparison, synthesis of a stationary phase-specific catalase, KatE, in E. coli is under the control of the alternative RNA polymerase sigma factor RpoS [12,13]. It is therefore tempting to speculate that such a regulatory system may also be present in AOB.

4.3 Regulation of enzyme synthesis during microcolony formation

Growth as microcolonies significantly reduced catalase specific activities in Ps. aeruginosa when compared to liquid-grown cells [14]. In comparison, catalase activities in N. europaea after surface growth (microcolony formation) on membrane filters were found to be elevated by over 50% (Table 1), but this appeared not to involve induction of new catalase isozymes. In contrast, the SOD activities found in N. europaea cells after surface growth were only 30% of those recorded in cells from batch culture growth. Although these changes were not excessive, their different direction (catalase increasing and SOD decreasing) indicated that surface growth was indeed affecting the oxidative stress response in N. europaea. One likely possibility is that cell density-dependent gene regulation caused the different response to oxidative stress observed in surface-grown compared to suspended AOB. In the biofilm of several cell layers, an important element of gene regulation could be the small homoserine lactone (HSL) molecules accumulating within the constrained intercellular spaces of biofilms. HSL molecules are already known to influence the recovery (growth initiation) of AOB after nutrient starvation [15], but as yet there is no direct evidence for a role of HSL-mediated gene regulation of the oxidative stress response in AOB. Interestingly, Ps. aeruginosa mutants in HSL synthesis are more sensitive to H₂O₂ damage than the wild-type [14]. An alternative explanation might be that cells grown in liquid are exposed to a different oxidising environment to those grown on surfaces, resulting in altered expression of stress enzymes or there could be direct effects of environmental factors on the enzymes themselves. Unfortunately, it was not possible to grow Nsp. multiformis on filters and no data are available on surface-induced changes of catalase and SOD activities in this organism. This was particularly unfortunate since Nsp. multiformis had already shown strong regulation of the enzymes during batch culture growth (Fig. 2B). In conclusion, AOB, as judged from the data obtained with N. europaea, may regulate their response to oxidative stress differently in biofilms as compared to pelagic environments and more than one strategy appears to have been adopted to modulate oxidative stress enzyme production.

Acknowledgements

This work was supported by Grant 9600638 from the Danish Agricultural and Veterinary Science Research Council. We thank James Moir for help during analysis of the N. europaea genome sequence database.

Author notes

Present address: Department of Molecular Biology and Biotechnology, University of Sheffeld, Firth Court, Western Bank, Sheffield S10 2TN, UK.