Di erential regulation of amoa and amob gene copies in Nitrosomonas europaea

FEMS Microbiology Letters 192 (2000) 163^168 www.fems-microbiology.org Di erential regulation of amoa and amob gene copies in Nitrosomonas europaea Lisa Y. Stein, Luis A. Sayavedra-Soto, Norman G. Hommes, Daniel J. Arp * Laboratory for Nitrogen Fixation Research, Department of Botany and Plant Pathology, Oregon State University, 2082 Cordley, Corvallis, OR 97331-2902, USA Received 13 June 2000; received in revised form 6 September 2000; accepted 7 September 2000 Abstract Nitrosomonas europaea contains two nearly identical copies of the operon, amocab, which encodes the ammonia monooxygenase (AMO) enzyme. Cells of N. europaea containing single mutations in either amoa or amob gene copies were incubated in ammonium both prior to and after exposure to acetylene or light. For each strain, the O 2 consumption rates and amounts of AmoA polypeptide, the active site-containing subunit of AMO, produced in each strain were determined. Strains carrying a mutation in either the amoa 2 or amob 2 genes responded similarly to wild-type cells, but the strains carrying mutations in the amoa 1 or amob 1 genes responded differently from the wildtype, or from each other. These results suggest that the copies of amoa and amob are differentially regulated upon exposure to different external stimuli. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords: Ammonia monooxygenase; Ammonia oxidation; Gene regulation; Multiple gene copy; Nitrosomonas europaea 1. Introduction Many strains of ammonia-oxidizing bacteria contain two or more nearly identical copies of the genes encoding the primary enzymes for ammonia oxidation ^ ammonia monooxygenase (AMO), hydroxylamine oxidoreductase, and two c-type cytochromes [1^6]. Together, these enzymes catalyze the oxidation of NH 3 to NO 3 2 via the formation of a NH 2 OH intermediate and thereby provide all of the energy required for the growth and maintenance of the cell. The AMO enzyme is encoded by three genes that are organized in the amocab operon. Two copies of the full operon are present in Nitrosomonas europaea, with an additional copy of amoc elsewhere in the genome [7]. The amoa gene encodes the active site-containing subunit of AMO, but the functions of the amob and amoc gene products are unknown. To study the function and necessity of each amoa gene copy to ammonia metabolism, disruption of expression by mutagenesis has been performed for the two amoa genes [8]. Both of the mutated strains were capable of cellular growth and division. How- * Corresponding author. Tel.: +1 (541) 737-4214; Fax: +1 (541) 737-3573; E-mail: arpd@bcc.orst.edu ever, in one of the strains (strain A1 in the present study), the growth rate decreased by 25%, and the production of amoa 2 mrna was about 63% that of the wild-type cells [8]. The strain containing the other amoa mutation (strain A2 in the present study) exhibited both a wild-type growth rate and amoa 1 mrna production. The rates of NH 4 - dependent O 2 consumption, a measure of AMO activity, mirrored the growth rates of the cultures, indicating a relationship between the amount of active AMO in the cell and the rapidity of cell growth and division. These results indicated that only one of the amoa gene copies, amoa 1, can fully compensate for the loss of expression of the other gene copy. An analogous genetic system to amocab exists for the particulate methane monooxygenase (pmmo), and was originally characterized in Methylococcus capsulatus Bath [9]. The gene order in the pmo operon is pmocab, the operon is present in two nearly identical copies, and there is an extra copy of pmoc within the genome [9,10]. In similar studies to those of N. europaea, strains of M. capsulatus Bath containing single pmoa, pmob or pmoc mutations were examined for their ability to grow on methane [10]. Similar to N. europaea, strains containing a mutation in one of the operons had a growth rate 15^ 20% slower than that of the wild-type, whereas strains with mutations in the other operon had nearly wild-type 0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S0378-1097(00)00426-2

164 L.Y. Stein et al. / FEMS Microbiology Letters 192 (2000) 163^168 growth rates. The pmmo activity, as measured by CH 4 - dependent O 2 consumption, was depressed in all of the strains relative to wild-type, but much more depressed in the slow-growing strains. The combined activity from expression of both pmocab copies was required to achieve full levels of pmmo activity [10]. We have shown previously that NH 3 is a strong regulator of amoa expression at both the transcriptional and translational levels [11,12]. When N. europaea cells were exposed to di erent concentrations of NH 3, by altering the ph of a NH 4 -containing medium, the AMO activity underwent a 2-fold increase over a 3-h period followed by a decrease to the initial level over the next 5 h [13]. The increase in activity was accompanied by the synthesis of both amoa mrna and AmoA polypeptides, suggesting that the creation of new AMO molecules was responsible for the increase in ammonia-oxidizing activity [13,14]. In the present study, we have determined how the expression di ers between the amocab 1 and amocab 2 operons when cells of N. europaea containing mutations in either the amoa or amob genes are exposed to di erent external stimuli. We incubated cells from four mutant strains and a wild-type strain of N. europaea in ammonium to stimulate the characterized increase in AMO activity. We compared the extent of the increase of activity, and the production of AmoA polypeptides to determine the relative contributions of each gene copy to the overall activity of AMO in the cell. In other experiments, we exposed cells of each strain to acetylene or light to destroy the AMO activity. Acetylene and light have been characterized as potent, irreversible, inactivators of AmoA [15]. The AMO activity was then recovered by incubation of the cells in ammonium, and we again examined the rates of activity increase and production of AmoA to determine the contributions of each gene copy to the total ammoniaoxidizing activity of the cells. 2. Materials and methods 2.1. Bacterial cultures Strains of N. europaea containing disrupted amoa or amob genes, as described elsewhere [8], were used. Brie y, the mutated strains of N. europaea (wild-type background ATCC 19178) have a gene cassette encoding kanamycin resistance inserted into BamHI restriction sites in the amoa 1, amoa 2, amob 1, and amob 2 genes. Cells of N. europaea (wild-type, A1, A2, B1, and B2) were grown in liquid batch cultures (1.5 l) on a rotary shaker (200 rpm) at 30³C in the dark. The de ned growth medium was as previously described [15]. The strains A1 and B1 required an extra inoculum of cells to ensure that maximum biomass production occurred within the same period for all of the cultures. Cells were harvested in late exponential phase by centrifugation (10 000Ug for 10 min), washed twice in sodium phosphate bu er (50 mm NaH 2 PO 4, 2 mm MgCl 2, at ph 8), then resuspended in 1.5 ml sodium phosphate bu er and kept at 4³C for 12^18 h to allow degradation of the amoa mrna [11]. Incubation at 4³C ensured that any increases in the ammonia-oxidizing activity observed during the experiments were a product of the translation of the newly synthesized amo mrna into new Amo protein. 2.2. Batch incubations and activity measurements All batch incubations of cells were conducted in growth medium containing 50 mm NH 4 as a substrate and 4 mm Na 2 CO 3 as a carbon source. Batch incubations were conducted in asks (125 ml) containing 25 ml medium and 200 Wl cell suspension (ca. 10 9 cells ml 31 ). The initial ph of the incubation medium was 8.0, and decreased after 8 h to about ph 6.5 and ph 6 for incubations conducted with and without prior exposure to acetylene or light, respectively. At the indicated times, 1 ml of the incubation mixture was removed, the cells were sedimented (14 000Ug for 2 min), and the supernatant was analyzed for NO 3 2 content [16] and ph. The cells were washed once and resuspended in sodium phosphate bu er (50 Wl). The cells were then used to determine the rate of NH 4 -dependent O 2 consumption using a Clark-style oxygen electrode (Yellow Springs Instruments Co., Yellow Springs, OH, USA) as described previously [15]. The total amount of cellular protein (20 mg ml 31 ) did not change throughout any of the incubations, suggesting that signi cant biomass production did not occur over the course of the experiments (data not shown). 2.3. Inactivation of AMO activity Cells to be inactivated by acetylene were resuspended in 25 ml sodium phosphate bu er in glass vials (160 ml) sealed with butyl rubber and aluminum crimp seals. Acetylene (1.6 ml) was added as overpressure to the vials and the mixture was incubated on a shaker (200 rpm) at 30³C for 1 h. Cells inactivated by light were prepared in glass vials as described above, and exposed to light from a slide projector for 1 h while shaking at a distance of 25 cm from the light source. The cells were washed with sodium phosphate bu er, added to incubations containing growth medium, and analyzed for NH 4 -dependent O 2 consumption activity. 2.4. Determination of AmoA polypeptide synthesis Cells from all ve strains were incubated as described above either before or after exposure to acetylene or light. The cells were incubated with medium (25 ml) in sealed vials (160 ml) containing 5 WCi Na 14 2 CO 3 (2^10 mci mmol 31, Sigma). After 3 or 4 h, cells were removed (1 ml), sedimented, and washed with sodium phosphate

L.Y. Stein et al. / FEMS Microbiology Letters 192 (2000) 163^168 165 bu er. The cells were resuspended in sodium dodecyl sulfate^polyacrylamide gel electrophoresis (SDS^PAGE) sample bu er (100 Wl) and kept at 380³C. Polypeptides were resolved and visualized on SDS^PAGE (12% acrylamide). Polypeptides that had incorporated 14 C from the xation of 14 CO 2 were visualized on a Phosphorimager screen and the amount of radiolabel was quanti ed with Imagequant software (Molecular Dynamics, Sunnyvale, CA, USA). 3. Results and discussion The NH 4 -dependent O 2 consumption rates were compared over an 8-h time course for ve strains of N. europaea containing single mutations in amoa (strains A1 and A2), amob (strains B1 and B2), or with no mutation (strain WT; Fig. 1). All ve strains underwent the characteristic increase in ammonia-oxidizing activity over the rst 3 h of the incubation, followed by a subsequent decrease, as controlled by the availability of NH 3 [13]. By Fig. 1. Time course of changes in the NH 4 -dependent O 2 consumption rate for strains of N. europaea incubated in growth medium. Washed cells were assayed for activity, as described in Section 2. Error bars represent the standard error for the average of three replicate experiments. The rate of O 2 consumption at 100% activity was de ned by the activity of each strain at time = 0 h. All strains had approximately the same activity at the beginning of each experiment; 100^120 nmol O 2 consumed min 31 ml 31. (A) Percent change in the NH 4 -dependent O 2 consumption rate is shown for the WT (b), A1 (F), and A2 (R) strains. (B) Percent change in the NH 4 -dependent O 2 consumption rate is shown for the WT (b), B1 (F), and B2 (R) strains. Fig. 2. Pools of radiolabeled AmoA polypeptides in ve strains of N. europaea after incubation for 3 h in ammonium. Cells from the WT, A1, A2, B1, and B2 strains were incubated in the presence of Na 14 2 CO 3 to radiolabel newly synthesized polypeptides as described in Section 2. The level of radiolabeled AmoA in each mutated strain was normalized to the amount of radiolabel present in the WT strain for each of three experiments. Thus, the average values and standard error for the A1, A2, B1 and B2 strains represent the deviation from the WT values. assigning the WT strain with a maximum activity increase of 100% (at 3 h), the A1 strain reached an average of 38%, the A2 71%, the B1 79%, and the B2 102% of the WT maximum activity increase. Based on the results of a twotailed Student's t-test, the di erences in the maximum activity increase among the A1, A2 and WT strains were statistically signi cant at the 95% con dence interval. The di erences among the B1, B2 and WT strains were not statistically signi cant. The production of NO 3 2 and concomitant decrease in ph by all of the strains over the 8-h time course were similar, with a slightly lower rate in the A1 strain (data not shown). Because di erent levels of activity were observed in each strain, we examined the quantities of AmoA polypeptides produced after 3 h, the time at which the maximal activity level was reached, to determine if the rate of ammonia oxidation activity was related to the quantity of AMO enzyme. To visualize newly made polypeptides, cells were incubated with media containing Na 14 2 CO 3. Because N. europaea is an obligate autotroph, it xes the 14 CO 2 and incorporates the radiolabeled carbon into newly synthesized proteins. The amount of radiolabel incorporated into the AmoA polypeptide (27 kda on SDS^PAGE) after a 3-h incubation in ammonium was set as 100% in the WT strain in each of three separate experiments (Fig. 2). Relative to the WT, the A1 strain incorporated an average of 50%, the A2 80%, the B1 48%, and the B2 74% of radiolabel into the AmoA polypeptide. The di erence in radiolabel incorporation between the A1 and A2 strains was statistically signi cant at the 90% con dence interval, but was not statistically signi cant between the B1 and B2 strains based on a Student's t-test. The combined NH 4 -dependent O 2 consumption activity of the A1 and A2 strains at 3 h was comparable to the full activity of the WT, which was similar to the results re-

166 L.Y. Stein et al. / FEMS Microbiology Letters 192 (2000) 163^168 Fig. 3. Time course for changes in the NH 4 -dependent O 2 consumption rate for strains of N. europaea incubated in ammonium for 8 h after exposure to either acetylene (A) or light (B) as described in Section 2. Activities are shown for the WT (b), A1 (F), and A2 (R) strains. The activity level at 100% was de ned in each strain as the amount of activity at time = 0 h for cells that were not exposed to acetylene or light (100^120 nmol O 2 min 31 ml 31 ). Error bars represent the standard error for the average of three replicate experiments. ported in M. capsulatus Bath cells containing pmoa mutations [10]. However, the ammonia-oxidizing activities of the B1 and B2 strains were similar to previous results with N. europaea strains containing amoa mutations, in which one of the strains had a modest decrease in activity relative to the other strain and the wild-type [8]. Discrepancies between the current results with the strains containing amoa mutations and the previously published results [8] are likely due to a di erence in the experimental conditions. In previous studies, the cells were actively growing, but no growth was detected in the incubations in the present study (data not shown). The amounts of newly synthesized AmoA polypeptides were equivalent in the A1 and B1 strains and in the A2 and B2 strains, but none of the strains synthesized a pool of AmoA that was equivalent to their observed rate of NH 4 -dependent O 2 consumption. The combined results suggest several layers of regulation for AMO, both at the translational and post-translational levels, upon exposure of cells to ammonia. First, the decreased production of AmoA polypeptides from the active operon in the A1 and B1 strains (amocab 2 ), relative to the A2 and B2 strains (amocab 1 ), suggests that the amocab operons were expressed di erently. Second, wild-type levels of ammonia-oxidizing activity were achieved in the B1 strain, but not in the A1 strain, indicating a di erence in signaling pathways after the AMO enzyme molecules were produced. It appears that the cells can compensate for the loss of amob expression from either operon, and from the loss of amoa 2 expression, but not from amoa 1 expression. One of the major di erences between strains with amoa versus amob mutations is that the expression of amob is expected to be deleted in these strains since the genes are co-transcribed [1,7]. The recovery of ammonia-oxidizing activity and production of AmoA polypeptides were also examined after exposure of cells to either acetylene or light, which specifically inactivate the AMO enzyme [15]. Regulation of the amocab operons should be quite di erent under these conditions relative to the above experiments, because here the cells are recovering from the inactivation of AMO rather than simply responding to ammonia. The Fig. 4. Experiments and reported results are as in Fig. 3. (A) Recovery of ammonia-oxidizing activity for cells exposed to acetylene. (B) Recovery of ammonia-oxidizing activity for cells exposed to light. Activities are shown for WT (b), B1 (F), and B2 (R) strains. Error bars represent the standard error for the average of three replicate experiments.

L.Y. Stein et al. / FEMS Microbiology Letters 192 (2000) 163^168 167 ve strains of N. europaea were incubated with 1% (v/v) acetylene or exposed to intense visible light. Both treatments inactivated the ammonia-oxidizing activity after a 1-h exposure. The ammonia-oxidizing activity was recovered over an 8-h period by incubating the cells in media containing ammonium. The AMO activity of cells from each strain that had not been exposed to an inactivator was set as 100%. Although there was no signi cant di erence among the experiments relative to untreated controls, the A1 and A2 strains consistently recovered their ammonia-oxidizing activity at the same rate or slightly faster than the WT after exposure to either acetylene or light (Fig. 3). After a 4-h incubation in ammonium, the A1 strain had an average of 104% and 112%, and the A2 116% and 129% activity during recovery from acetylene or light exposure, respectively. The rate of NO 3 2 production by each of the strains was equivalent (data not shown). However, the recovery of activity in the B1 strain was consistently slower than in the B2 or WT strains in both the acetylene and light treatments (Fig. 4). After 4 h, the B1 strain had an average of 82% and 76% of the B2 and WT activity during recovery from acetylene or light, respectively. The di erence in activity between the B1 and the other two strains during recovery from acetylene was signi cant at a 90% con dence interval (Student's t-test), but the di erence in activity among the strains during recovery from light was not statistically signi cant. Also, the activity in the B1 strain never reached the level of activity observed in untreated cells in every experiment performed. To regain ammonia-oxidizing activity after exposure to acetylene or light, the cells must synthesize new polypeptides [15]. Production of AmoA polypeptides was examined after 4 h of recovery in all of the strains (Fig. 5). The amount of AmoA synthesized after 4 h in the WT strain was set as 100% for each of three experiments. Again, although there was variability between experiments, the trends of relative polypeptide synthesis within each strain were consistent. During recovery from acetylene exposure, the A1 strain reached an average of 88%, the A2 strain 93%, the B1 strain 37% and the B2 strain 101% of the WT level of AmoA polypeptides(fig. 5A). There was a signi cant di erence in polypeptide levels between the B1 and B2 strains at a con dence interval of 90%, based on a Student's t-test. During the recovery from light exposure, the A1 strain reached an average of 90%, the A2 strain 117%, and B1 strain 96%, and the B2 strain 139% of the WT level of AmoA polypeptides (Fig. 5B). The di erences among these values were not statistically signi cant. These results suggest that the level of AmoA polypeptide synthesis required to recover the ammonia-oxidizing activity was dependent both on the mechanism of AMO inactivation and on the genetic background of the cells. In the A1 and A2 strains, there was a reasonable correlation between the amount of AmoA synthesized and the amount of ammonia-oxidizing activity recovered. These Fig. 5. Pools of radiolabeled AmoA polypeptides in ve strains of N. europaea after inactivation by acetylene (A) or light (B) followed by incubation in ammonium for 4 h. Cells were treated similarly to that described in Fig. 2. The level of radiolabeled AmoA in each mutated strain was normalized to the amount of radiolabel present in the WT strain for each of three experiments. Thus, the average values and standard error for the A1, A2, B1 and B2 strains represent the deviation from the WT values. results imply that under conditions where AMO activity is recovering from prior inactivation, either of the amo- CAB operons can compensate for the loss of amoab expression in the other operon. However, in the B1 strain, there was a large depression of AmoA synthesis upon exposure to acetylene, but not upon exposure to light. The cells were able to compensate for this di erence in AmoA pools, however, and most of the ammonia-oxidizing activity was regained. In the B2 strain, there was a correlation between AmoA synthesis and recovery of ammonia-oxidizing activity after exposure to acetylene, but more AmoA had to be synthesized to regain activity during recovery from light exposure. The results from the B1 and B2 strains suggest that separate mechanisms exist for the recovery of AMO following acetylene or light exposure, and that the cells were less able to recover their full level of activity only when the amob 1 gene was inactivated. Taking the results together, there appears to be a complex set of responses engaged by cells of N. europaea following inactivation of AMO that rely on the presence and activity of speci c sets of genes. This study shows that the two copies of the key metabolic genes, amoa and amob, are di erentially regulated in

168 L.Y. Stein et al. / FEMS Microbiology Letters 192 (2000) 163^168 N. europaea. The di erent expression patterns of AmoA polypeptides in all of the strains studied suggest that the cell maintains two nearly identical copies of the amocab operon in order to mount a quick and speci c response to external stimuli. The controlling factor(s) for the expression of the amo genes is apparently not within the coding sequence, implying that there are potential regions either upstream or downstream of the operons that can control transcription of these genes. Although we are continuing to search for such controlling regions, sequences surrounding amocab have not yielded any signi cant insights thus far. Beyond the control over amocab transcription, this study also revealed the complexity behind the regulation of AMO activity. Our results suggest that the cell has multiple layers of regulation at the translational and post-translational levels that allow the cell to maintain a level of ammonia-oxidizing activity that is in concert with its environment. Acknowledgements This work was supported by DOE Grant DE-FG03-97ER20266 to D.J.A. and L.A.S.-S. References [1] McTavish, H., Fuchs, J.A. and Hooper, A.B. (1993a) Sequence of the gene coding for ammonia monooxygenase in Nitrosomonas europaea. J. Bacteriol. 175, 2436^2444. [2] McTavish, H., LaQuier, F., Arciero, D., Logan, M., Mundfrom, G., Fuchs, J.A. and Hooper, A.B. (1993b) Multiple copies of genes coding for electron transport proteins in the bacterium Nitrosomonas europaea. J. Bacteriol. 175, 2445^2447. [3] Norton, J.M., Jackie, M.L. and Klotz, M.G. (1996) The gene encoding ammonia monooxygenase subunit A exists in three nearly identical copies in Nitrosospira sp. NpAV. FEMS Microbiol. Lett. 139, 181^188. [4] Klotz, M.G. and Norton, J.M. (1997) A gene encoding a membrane protein exists upstream of the amoa/amob genes in ammonia oxidizing bacteria; a third member of the amo operon? FEMS Microbiol. Lett. 150, 65^73. [5] Sayavedra-Soto, L.A., Hommes, N.G. and Arp, D.J. (1994) Characterization of the gene encoding hydroxylamine oxidoreductase in Nitrosomonas europaea. J. Bacteriol. 176, 504^510. [6] Bergmann, D.J., Arciero, D.M. and Hooper, A.B. (1994a) Organization of the hao gene cluster of Nitrosomonas europaea: genes for two tetraheme c cytochromes. J. Bacteriol. 1767, 3148^3153. [7] Sayavedra-Soto, L.A., Hommes, N.G., Arp, D.J., Alzerreca, J., Norton, J.M. and Klotz, M.G. (1998) Transcription of amoc, amoa and amob genes in Nitrosomonas europaea and Nitrosospira sp. NpAV. FEMS Microbiol. Lett. 167, 81^88. [8] Hommes, N.G., Sayavedra-Soto, L.A. and Arp, D.J. (1998) Mutagenesis and expression of amo, which codes for ammonia monooxygenase in Nitrosomonas europaea. J. Bacteriol. 180, 3353^3359. [9] Semrau, J.D., Chistoserdov, A., Lebron, J., Costello, A., Davagnino, J., Kenna, E., Holmes, A.J., Finch, R., Murrell, J.C. and Lidstrom, M.E. (1995) Particulate methane monooxygenase genes in methanotrophs. J. Bacteriol. 177, 3071^3079. [10] Stolyar, S., Costello, A.M., Peeples, T.L. and Lidstrom, M.E. (1999) Role of multiple gene copies in particulate methane monooxygenase activity in the methane-oxidizing bacterium Methylococcus capsulatus Bath. Microbiology 145, 1235^1244. [11] Sayavedra-Soto, L.A., Hommes, N.G., Russell, S.A. and Arp, D.J. (1996) Induction of ammonia monooxygenase and hydroxylamine oxidoreductase mrnas by ammonium in Nitrosomonas europaea. Mol. Microbiol. 20, 541^548. [12] Hyman, M.R. and Arp, D.J. (1995) E ects of ammonia on the de novo synthesis of polypeptides in cells of Nitrosomonas europaea denied ammonia as an energy source. J. Bacteriol. 177, 4974^4979. [13] Stein, L.Y., Arp, D.J. and Hyman, M.R. (1997) Regulation of the synthesis and activity of ammonia monooxygenase in Nitrosomonas europaea by altering ph to a ect NH 3 availability. Appl. Environ. Microbiol. 63, 4588^4592. [14] Stein, L.Y. and Arp, D.J. (1998) Ammonium limitation results in the loss of ammonia-oxidizing activity in Nitrosomonas europaea. Appl. Environ. Microbiol. 64, 1514^1521. [15] Hyman, M.R. and Arp, D.J. (1992) 14 C 2 H 2 - and 14 CO 2 -labeling studies of the de novo synthesis of polypeptides by Nitrosomonas europaea during recovery from acetylene and light inactivation of ammonia monooxygenase. J. Biol. Chem. 267, 1534^1545. [16] Hageman, R.H. and Hucklesby, D.P. (1971) Nitrate reductase in higher plants. Methods Enzymol. 23, 491^503.