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Catalase and superoxide dismutase activity in ammonia-oxidising bacteria
FEMS Microbiology Ecology 38 (2001) 53^58
www.fems-microbiology.org
Catalase and superoxide dismutase activity in ammonia-oxidising bacteria Nicholas J. Wood 1 , Jan SÖrensen * Section of Genetics and Microbiology, Department of Ecology, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark Received 19 June 2001; received in revised form 5 September 2001; accepted 5 September 2001 First published online 24 October 2001
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. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Ammonia-oxidizing bacterium; Catalase; Superoxide dismutase; Oxidative stress ; Bio¢lm
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
* Corresponding author. Tel.: +45 35 28 26 26; Fax: +45 35 28 26 06. E-mail address : [email protected] (J. SÖrensen). 1 Present address: Department of Molecular Biology and Biotechnology, University of She¤eld, Firth Court, Western Bank, She¤eld S10 2TN, UK.
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. Speci¢c 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 runo¡ waters. These ¢ndings 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,
0168-6496 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 6 4 9 6 ( 0 1 ) 0 0 1 7 3 - 8
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which have been adopted to cope with ROS in these bacteria. Substantial di¡erences in both the speci¢c activities of enzymes and their regulation between the di¡erent AOB were observed. The results present the ¢rst demonstration of oxidative stress enzymes, including di¡erent patterns of regulation, in AOB.
fragments were removed and the lysate was concentrated by centrifugation at 4³C in a Microcon 10 concentrator (10 kDa MW cuto¡; Millipore) resulting in a ¢nal volume of approximately 50 Wl. Total protein concentration in cell lysates was determined by a modi¢cation of the Lowry procedure (Sigma Kit P-5656) using bovine serum albumin as the standard.
2. Materials and methods
2.3. Spectrophotometric catalase and SOD assays
2.1. Bacterial strains and culture conditions
The catalase and SOD speci¢c activities of cell lysates were determined using the spectrophotometric protocols given in [6]. One unit of catalase was de¢ned as the amount of lysate that catalysed the decomposition of 1 Wmol H2 O2 per min at 25³C. One unit of SOD inhibited the rate of xanthine/xanthine oxidase-dependent cytochrome c reduction at 25³C by 50%.
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 500ml 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 37³C. Pseudomonas £uorescens DR54 [5] was grown in the same medium but incubated at 30³C. In some experiments oxidative stress was applied to early stationary phase cells by addition of 40 WM hydrogen peroxide (¢nal 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 ¢lters (0.2 Wm 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 Wm2 of ¢lter surface. Filters were then £oated on 5 ml of the appropriate medium for 5 days at 30³C in the dark.
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 1UTris-glycine bu¡er, 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
2.2. Cell harvest and lysis procedures
3.1. Catalase and SOD enzymes in AOB strains
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 bu¡er (50 mM phosphate, pH 7.8). After re-centrifugation the pellets were stored at 320³C. Prior to cell lysis, frozen pellets were resuspended in 50 Wl phosphate bu¡er and then subjected to ¢ve 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 4³C the lysate was ready for use. Filter-grown cells were washed once by £otation of the ¢lters on the surface of 5 ml ice-cold potassium phosphate bu¡er with gentle shaking for 5 min and the ¢lters were then frozen at 320³C until required. When lysis was performed, ¢lters (usually six) were cut into approximately 5mm square pieces with scissors and `resuspended' in 400 Wl phosphate bu¡er. Following sonication as above the ¢lter
In order to obtain su¤cient cell mass, extracts were prepared initially from cells harvested at early stationary phase in liquid batch culture. To compare the results from nitri¢er cells with more commonly described organisms, extracts were also prepared from cells of Ps. aeruginosa, Ps. £uorescens and E. coli. As shown in Table 1, all the AOB tested possessed catalase activity. N. europaea possessed comparatively high speci¢c 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. £uorescens strain possessed intermediate activity while E. coli had speci¢c activities that were similar to Nsp. multiformis and Nitrosospira AV. Table 1 also shows that all AOB possessed SOD activity. Speci¢c activities of N. europaea SOD were similar to those of the other nitri¢ers, with Nitrosospira
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AV possessing the highest SOD activity. Ps. aeruginosa had the highest SOD activity of all the organisms tested, while activities in Ps. £uorescens, E. coli and AOB were in the same range. The isozyme pro¢les 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. £uorescens and E. coli all possessed only one zone of SOD activity under the conditions employed (Fig. 1B). 3.2. Regulation of catalase and SOD enzyme production ^ e¡ect of growth cycle in AOB batch cultures The above comparison was based on cells harvested in early stationary phase. To determine whether speci¢c activities and isozyme banding patterns varied with growth phase, cell extracts were prepared from N. europaea and Nsp. multiformis cultures harvested at di¡erent points throughout the growth cycle. The regulation of catalase and SOD production di¡ered in the two organisms (Fig. 2). N. europaea produced catalase and SOD at only slightly (max. 2-fold) lower speci¢c 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 speci¢c activities rapidly fell from a high speci¢c activity in the inoculum to a low value of approximately 10^20 units mg31 protein in the exponenTable 1 Oxidative stress enzymes in ammonia-oxidising bacteria and some other organisms Organism N. europaeaa N. europaeab Nsp. multiformisa Nitrosospira Apple Valleya Ps. aeruginosaa Ps. £uorescensa E. colia
Enzyme activity (units (mg protein)31 ) Catalase
SOD
1535 þ 75 2504 þ 346 66 þ 9 62 þ 14 1335 þ 283 253 þ 38 60 þ 12
71 þ 3 22 þ 6 88 þ 8 130 þ 9 193 þ 37 67 þ 7 42 þ 1
Results are presented as the standard error of the mean of at least triplicate samples. a Liquid-grown cells from 20-ml batch cultures in 50-ml screw cap tubes, harvested in early stationary phase. b Surface-grown cells from membrane ¢lters, harvested after bio¢lm formation.
Fig. 1. Catalase and SOD isozyme pro¢les 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. £uorescens. The ¢gure is a composite image of several gels but the relative migration rates of the isozymes are comparable.
tially 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 speci¢c activities after the culture had been growtharrested for some time. However, the increase in SOD speci¢c activity was less pronounced than that of catalase in stationary phase; the SOD speci¢c activities increased approximately 4-fold when comparing low and high points during the whole growth cycle. The constant catalase speci¢c activity in N. europaea corresponded well with the observation of only a single
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Fig. 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 (F). At various times the cultures were sacri¢ced and cells harvested for later estimation of catalase (b) and SOD (R) activity. Error bars represent standard errors of the mean from at least three replicate cultures. Lanes 1^5 show the catalase isozyme pro¢les 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 di¡erent catalase isozymes in Nsp. multiformis, as discussed in the text.
catalase isozyme throughout the growth cycle (Fig. 2C). Similarly, the constant SOD speci¢c 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. E¡ect of colony growth on regulation of catalase and SOD enzyme production Previously, it has been observed that growth on surfaces
may a¡ect AOB tolerance to stress (osmotic pressure) [4] so in the present study the e¡ect 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 ¢lters were extracted and compared to those of liquid-grown cultures. During incubation the inoculated cells formed microcolonies, and after 5 days a nearly con£uent layer of AOB cells formed a thin bio¢lm on the ¢lters. As shown in Table 1, surfacegrown cells produced 2-fold higher and 5-fold lower speci¢c activities of catalase and SOD respectively. However, PAGE analysis of extracts of ¢lter-grown cells, followed by staining for the catalase and SOD isozyme pro¢les, generated banding patterns similar to those of liquidgrown cells in batch cultures (data not shown). Hence, in N. europaea neither growth phase (liquid cultures) nor surface growth (bio¢lm cultures) seemed to regulate isozyme expression patterns. Unfortunately, it was not pos-
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As suggested above, the changes of enzyme speci¢c activities in liquid-grown and surface-grown cells could be due to di¡erent expression levels or direct e¡ects on the enzymes formed. To test the e¡ect of reactive oxygen species (exogenously supplied H2 O2 ) on the catalase and SOD speci¢c activities and isozyme patterns, cells of N. europaea and Nsp. multiformis were grown to late exponential phase and then supplemented with 40 WM H2 O2 for 4 h. After H2 O2 exposure, no signi¢cant di¡erences in either catalase or SOD speci¢c activities were found (data not shown). In addition, H2 O2 treatment elicited no change to isozyme banding patterns as determined by PAGE (data not shown).
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 modi¢cation.
4. Discussion
4.2. Regulation of enzyme synthesis during liquid batch culture
sible to culture Nsp. multiformis to an adequate cell density on the membrane surfaces. 3.4. E¡ects of reactive oxygen species (H2 O2 ) on catalase and SOD speci¢c activities
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 £uctuating 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 di¡erent 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 speci¢c 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 ¢rst 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 Q-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
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 speci¢c induction when the cultures enter stationary phase. By comparison, synthesis of a stationary phase-speci¢c 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.
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4.3. Regulation of enzyme synthesis during microcolony formation Growth as microcolonies signi¢cantly reduced catalase speci¢c 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 ¢lters 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 di¡erent direction (catalase increasing and SOD decreasing) indicated that surface growth was indeed a¡ecting 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 surfacegrown compared to suspended AOB. In the bio¢lm 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 bio¢lms. HSL molecules are already known to in£uence 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 H2 O2 damage than the wild-type [14]. An alternative explanation might be that cells grown in liquid are exposed to a di¡erent oxidising environment to those grown on surfaces, resulting in altered expression of stress enzymes or there could be direct e¡ects of environmental factors on the enzymes themselves. Unfortunately, it was not possible to grow Nsp. multiformis on ¢lters 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 di¡erently in bio¢lms as compared to pelagic environments and more than one strategy appears to have been adopted to modulate oxidative stress enzyme production. Acknowledgements
Danish Agricultural and Veterinary Science Research Council. We thank James Moir for help during analysis of the N. europaea genome sequence database. References [1] Bedard, C. and Knowles, R. (1989) Physiology, biochemistry, and speci¢c inhibitors of CH4 , NH 4 and CO oxidation by methanotrophs and nitri¢ers. Microbiol. Rev. 53, 68^84. [2] Iizumi, T. and Nakamura, K. (1997) Cloning, nucleotide sequence, and regulatory analysis of the Nitrosomonas europaea dnaK gene. Appl. Environ. Microbiol. 63, 1777^1784. [3] Draper, W.M. and Crosby, D.G. (1983) The photochemical generation of hydrogen peroxide in natural waters. Arch. Environ. Contam. Toxicol. 12, 121^126. [4] Wood, N.J. and SÖrensen, J. (1998) Osmotic stimulation of microcolony formation in Nitrosomonas europaea. FEMS Microbiol. Ecol. 27, 175^183. [5] Nielsen, M.N., SÖrensen, J., Fels, J. and Pedersen, H.C. (1998) Secondary metabolite- and endochitinase-dependent antagonism toward plant-pathogenic microfungi of Pseudomonas £uorescens isolates from sugar beet rhizosphere. Appl. Environ. Microbiol. 64, 3563^3569. [6] Katsuwon, J. and Anderson, A.J. (1989) Response of plant-colonizing Pseudomonads to hydrogen peroxide. Appl. Environ. Microbiol. 55, 2985^2989. [7] Woodbury, W., Spencer, A.K. and Stahmann, M.A. (1971) An improved procedure using ferricyanide for detecting catalase isozymes. Anal. Biochem. 44, 301^305. [8] Salin, M.L. and McCord, J.M. (1974) Superoxide dismutases in polymorphonuclear leukocytes. J. Clin. Invest. 54, 1005^1009. [9] Farr, S. and Kogoma, T. (1991) Oxidative stress responses in Escherichia coli and Salmonella typhimurium. Microbiol. Rev. 55, 561^585. [10] Kargalioglu, Y. and Imlay, J.A. (1994) Importance of anaerobic superoxide dismutase synthesis in facilitating outgrowth of Escherichia coli upon entry into an aerobic habitat. J. Bacteriol. 176, 7653^ 7658. [11] http://www.jgi.doe.gov/JGI_microbial/html/nitrosomonas_homepage. html [12] Loewen, P.C., Triggs, B.L., George, C.S. and Hrabauchuk, C.E. (1984) Genetic mapping of katF, a locus that with katE a¡ects the synthesis of a second catalase species in Escherichia coli. J. Bacteriol. 160, 669^675. [13] Mulvey, M.R., Switala, J., Borys, A. and Loewen, P.C. (1990) Regulation of transcription of katE and katF in Escherichia coli. J. Bacteriol. 172, 6713^6720. [14] Hassett, D.J., Ma, J.F., Elkins, J.G., McDermott, T.R., Ochsner, U.A., West, S.E.H., Huang, C.T., Frederiks, J., Burnett, S., Stewart, P.S., McFeters, G., Passador, L. and Iglewski, B.H. (1999) Quorum sensing in Pseudomonas aeruginosa controls expression of catalase and superoxide dismutase genes and mediates bio¢lm susceptibility to hydrogen peroxide. Mol. Microbiol. 34, 1082^1093. [15] Batchelor, S.E., Cooper, M., Chhabra, S.R., Glover, L.A., Stewart, G.S.A.B., Williams, P. and Prosser, J.I. (1997) Cell-density regulated recovery of starved bio¢lm populations of ammonia oxidising bacteria. Appl. Environ. Microbiol. 63, 2281^2286.
This work was supported by Grant 9600638 from the
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Catalase and superoxide dismutase activity in ammonia-oxidising bacteria Nicholas J. Wood 1 , Jan SÖrensen * Section of Genetics and Microbiology, Department of Ecology, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark Received 19 June 2001; received in revised form 5 September 2001; accepted 5 September 2001 First published online 24 October 2001
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. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Ammonia-oxidizing bacterium; Catalase; Superoxide dismutase; Oxidative stress ; Bio¢lm
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
* Corresponding author. Tel.: +45 35 28 26 26; Fax: +45 35 28 26 06. E-mail address : [email protected] (J. SÖrensen). 1 Present address: Department of Molecular Biology and Biotechnology, University of She¤eld, Firth Court, Western Bank, She¤eld S10 2TN, UK.
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. Speci¢c 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 runo¡ waters. These ¢ndings 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,
0168-6496 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 6 4 9 6 ( 0 1 ) 0 0 1 7 3 - 8
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which have been adopted to cope with ROS in these bacteria. Substantial di¡erences in both the speci¢c activities of enzymes and their regulation between the di¡erent AOB were observed. The results present the ¢rst demonstration of oxidative stress enzymes, including di¡erent patterns of regulation, in AOB.
fragments were removed and the lysate was concentrated by centrifugation at 4³C in a Microcon 10 concentrator (10 kDa MW cuto¡; Millipore) resulting in a ¢nal volume of approximately 50 Wl. Total protein concentration in cell lysates was determined by a modi¢cation of the Lowry procedure (Sigma Kit P-5656) using bovine serum albumin as the standard.
2. Materials and methods
2.3. Spectrophotometric catalase and SOD assays
2.1. Bacterial strains and culture conditions
The catalase and SOD speci¢c activities of cell lysates were determined using the spectrophotometric protocols given in [6]. One unit of catalase was de¢ned as the amount of lysate that catalysed the decomposition of 1 Wmol H2 O2 per min at 25³C. One unit of SOD inhibited the rate of xanthine/xanthine oxidase-dependent cytochrome c reduction at 25³C by 50%.
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 500ml 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 37³C. Pseudomonas £uorescens DR54 [5] was grown in the same medium but incubated at 30³C. In some experiments oxidative stress was applied to early stationary phase cells by addition of 40 WM hydrogen peroxide (¢nal 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 ¢lters (0.2 Wm 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 Wm2 of ¢lter surface. Filters were then £oated on 5 ml of the appropriate medium for 5 days at 30³C in the dark.
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 1UTris-glycine bu¡er, 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
2.2. Cell harvest and lysis procedures
3.1. Catalase and SOD enzymes in AOB strains
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 bu¡er (50 mM phosphate, pH 7.8). After re-centrifugation the pellets were stored at 320³C. Prior to cell lysis, frozen pellets were resuspended in 50 Wl phosphate bu¡er and then subjected to ¢ve 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 4³C the lysate was ready for use. Filter-grown cells were washed once by £otation of the ¢lters on the surface of 5 ml ice-cold potassium phosphate bu¡er with gentle shaking for 5 min and the ¢lters were then frozen at 320³C until required. When lysis was performed, ¢lters (usually six) were cut into approximately 5mm square pieces with scissors and `resuspended' in 400 Wl phosphate bu¡er. Following sonication as above the ¢lter
In order to obtain su¤cient cell mass, extracts were prepared initially from cells harvested at early stationary phase in liquid batch culture. To compare the results from nitri¢er cells with more commonly described organisms, extracts were also prepared from cells of Ps. aeruginosa, Ps. £uorescens and E. coli. As shown in Table 1, all the AOB tested possessed catalase activity. N. europaea possessed comparatively high speci¢c 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. £uorescens strain possessed intermediate activity while E. coli had speci¢c activities that were similar to Nsp. multiformis and Nitrosospira AV. Table 1 also shows that all AOB possessed SOD activity. Speci¢c activities of N. europaea SOD were similar to those of the other nitri¢ers, with Nitrosospira
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AV possessing the highest SOD activity. Ps. aeruginosa had the highest SOD activity of all the organisms tested, while activities in Ps. £uorescens, E. coli and AOB were in the same range. The isozyme pro¢les 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. £uorescens and E. coli all possessed only one zone of SOD activity under the conditions employed (Fig. 1B). 3.2. Regulation of catalase and SOD enzyme production ^ e¡ect of growth cycle in AOB batch cultures The above comparison was based on cells harvested in early stationary phase. To determine whether speci¢c activities and isozyme banding patterns varied with growth phase, cell extracts were prepared from N. europaea and Nsp. multiformis cultures harvested at di¡erent points throughout the growth cycle. The regulation of catalase and SOD production di¡ered in the two organisms (Fig. 2). N. europaea produced catalase and SOD at only slightly (max. 2-fold) lower speci¢c 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 speci¢c activities rapidly fell from a high speci¢c activity in the inoculum to a low value of approximately 10^20 units mg31 protein in the exponenTable 1 Oxidative stress enzymes in ammonia-oxidising bacteria and some other organisms Organism N. europaeaa N. europaeab Nsp. multiformisa Nitrosospira Apple Valleya Ps. aeruginosaa Ps. £uorescensa E. colia
Enzyme activity (units (mg protein)31 ) Catalase
SOD
1535 þ 75 2504 þ 346 66 þ 9 62 þ 14 1335 þ 283 253 þ 38 60 þ 12
71 þ 3 22 þ 6 88 þ 8 130 þ 9 193 þ 37 67 þ 7 42 þ 1
Results are presented as the standard error of the mean of at least triplicate samples. a Liquid-grown cells from 20-ml batch cultures in 50-ml screw cap tubes, harvested in early stationary phase. b Surface-grown cells from membrane ¢lters, harvested after bio¢lm formation.
Fig. 1. Catalase and SOD isozyme pro¢les 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. £uorescens. The ¢gure is a composite image of several gels but the relative migration rates of the isozymes are comparable.
tially 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 speci¢c activities after the culture had been growtharrested for some time. However, the increase in SOD speci¢c activity was less pronounced than that of catalase in stationary phase; the SOD speci¢c activities increased approximately 4-fold when comparing low and high points during the whole growth cycle. The constant catalase speci¢c activity in N. europaea corresponded well with the observation of only a single
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Fig. 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 (F). At various times the cultures were sacri¢ced and cells harvested for later estimation of catalase (b) and SOD (R) activity. Error bars represent standard errors of the mean from at least three replicate cultures. Lanes 1^5 show the catalase isozyme pro¢les 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 di¡erent catalase isozymes in Nsp. multiformis, as discussed in the text.
catalase isozyme throughout the growth cycle (Fig. 2C). Similarly, the constant SOD speci¢c 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. E¡ect of colony growth on regulation of catalase and SOD enzyme production Previously, it has been observed that growth on surfaces
may a¡ect AOB tolerance to stress (osmotic pressure) [4] so in the present study the e¡ect 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 ¢lters were extracted and compared to those of liquid-grown cultures. During incubation the inoculated cells formed microcolonies, and after 5 days a nearly con£uent layer of AOB cells formed a thin bio¢lm on the ¢lters. As shown in Table 1, surfacegrown cells produced 2-fold higher and 5-fold lower speci¢c activities of catalase and SOD respectively. However, PAGE analysis of extracts of ¢lter-grown cells, followed by staining for the catalase and SOD isozyme pro¢les, generated banding patterns similar to those of liquidgrown cells in batch cultures (data not shown). Hence, in N. europaea neither growth phase (liquid cultures) nor surface growth (bio¢lm cultures) seemed to regulate isozyme expression patterns. Unfortunately, it was not pos-
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As suggested above, the changes of enzyme speci¢c activities in liquid-grown and surface-grown cells could be due to di¡erent expression levels or direct e¡ects on the enzymes formed. To test the e¡ect of reactive oxygen species (exogenously supplied H2 O2 ) on the catalase and SOD speci¢c activities and isozyme patterns, cells of N. europaea and Nsp. multiformis were grown to late exponential phase and then supplemented with 40 WM H2 O2 for 4 h. After H2 O2 exposure, no signi¢cant di¡erences in either catalase or SOD speci¢c activities were found (data not shown). In addition, H2 O2 treatment elicited no change to isozyme banding patterns as determined by PAGE (data not shown).
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 modi¢cation.
4. Discussion
4.2. Regulation of enzyme synthesis during liquid batch culture
sible to culture Nsp. multiformis to an adequate cell density on the membrane surfaces. 3.4. E¡ects of reactive oxygen species (H2 O2 ) on catalase and SOD speci¢c activities
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 £uctuating 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 di¡erent 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 speci¢c 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 ¢rst 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 Q-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
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 speci¢c induction when the cultures enter stationary phase. By comparison, synthesis of a stationary phase-speci¢c 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.
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4.3. Regulation of enzyme synthesis during microcolony formation Growth as microcolonies signi¢cantly reduced catalase speci¢c 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 ¢lters 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 di¡erent direction (catalase increasing and SOD decreasing) indicated that surface growth was indeed a¡ecting 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 surfacegrown compared to suspended AOB. In the bio¢lm 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 bio¢lms. HSL molecules are already known to in£uence 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 H2 O2 damage than the wild-type [14]. An alternative explanation might be that cells grown in liquid are exposed to a di¡erent oxidising environment to those grown on surfaces, resulting in altered expression of stress enzymes or there could be direct e¡ects of environmental factors on the enzymes themselves. Unfortunately, it was not possible to grow Nsp. multiformis on ¢lters 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 di¡erently in bio¢lms as compared to pelagic environments and more than one strategy appears to have been adopted to modulate oxidative stress enzyme production. Acknowledgements
Danish Agricultural and Veterinary Science Research Council. We thank James Moir for help during analysis of the N. europaea genome sequence database. References [1] Bedard, C. and Knowles, R. (1989) Physiology, biochemistry, and speci¢c inhibitors of CH4 , NH 4 and CO oxidation by methanotrophs and nitri¢ers. Microbiol. Rev. 53, 68^84. [2] Iizumi, T. and Nakamura, K. (1997) Cloning, nucleotide sequence, and regulatory analysis of the Nitrosomonas europaea dnaK gene. Appl. Environ. Microbiol. 63, 1777^1784. [3] Draper, W.M. and Crosby, D.G. (1983) The photochemical generation of hydrogen peroxide in natural waters. Arch. Environ. Contam. Toxicol. 12, 121^126. [4] Wood, N.J. and SÖrensen, J. (1998) Osmotic stimulation of microcolony formation in Nitrosomonas europaea. FEMS Microbiol. Ecol. 27, 175^183. [5] Nielsen, M.N., SÖrensen, J., Fels, J. and Pedersen, H.C. (1998) Secondary metabolite- and endochitinase-dependent antagonism toward plant-pathogenic microfungi of Pseudomonas £uorescens isolates from sugar beet rhizosphere. Appl. Environ. Microbiol. 64, 3563^3569. [6] Katsuwon, J. and Anderson, A.J. (1989) Response of plant-colonizing Pseudomonads to hydrogen peroxide. Appl. Environ. Microbiol. 55, 2985^2989. [7] Woodbury, W., Spencer, A.K. and Stahmann, M.A. (1971) An improved procedure using ferricyanide for detecting catalase isozymes. Anal. Biochem. 44, 301^305. [8] Salin, M.L. and McCord, J.M. (1974) Superoxide dismutases in polymorphonuclear leukocytes. J. Clin. Invest. 54, 1005^1009. [9] Farr, S. and Kogoma, T. (1991) Oxidative stress responses in Escherichia coli and Salmonella typhimurium. Microbiol. Rev. 55, 561^585. [10] Kargalioglu, Y. and Imlay, J.A. (1994) Importance of anaerobic superoxide dismutase synthesis in facilitating outgrowth of Escherichia coli upon entry into an aerobic habitat. J. Bacteriol. 176, 7653^ 7658. [11] http://www.jgi.doe.gov/JGI_microbial/html/nitrosomonas_homepage. html [12] Loewen, P.C., Triggs, B.L., George, C.S. and Hrabauchuk, C.E. (1984) Genetic mapping of katF, a locus that with katE a¡ects the synthesis of a second catalase species in Escherichia coli. J. Bacteriol. 160, 669^675. [13] Mulvey, M.R., Switala, J., Borys, A. and Loewen, P.C. (1990) Regulation of transcription of katE and katF in Escherichia coli. J. Bacteriol. 172, 6713^6720. [14] Hassett, D.J., Ma, J.F., Elkins, J.G., McDermott, T.R., Ochsner, U.A., West, S.E.H., Huang, C.T., Frederiks, J., Burnett, S., Stewart, P.S., McFeters, G., Passador, L. and Iglewski, B.H. (1999) Quorum sensing in Pseudomonas aeruginosa controls expression of catalase and superoxide dismutase genes and mediates bio¢lm susceptibility to hydrogen peroxide. Mol. Microbiol. 34, 1082^1093. [15] Batchelor, S.E., Cooper, M., Chhabra, S.R., Glover, L.A., Stewart, G.S.A.B., Williams, P. and Prosser, J.I. (1997) Cell-density regulated recovery of starved bio¢lm populations of ammonia oxidising bacteria. Appl. Environ. Microbiol. 63, 2281^2286.
This work was supported by Grant 9600638 from the
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