Department of Biochemistry,1 Molecular and Cellular Biology Program, University of Washington, Seattle, Washington 981952
Received 23 December 2005/ Returned for modification 24 January 2006/ Accepted 14 April 2006
| ABSTRACT |
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| INTRODUCTION |
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Mutations within the LMNA gene cause a variety of human disorders collectively known as laminopathies. These include progeria syndromes and dystrophies associated with skeletal muscle and adipose tissue (5, 10, 15, 19, 45, 58). Mice lacking A-type lamins or deficient in processing lamin A develop skeletal and cardiac muscular dystrophies, thus confirming the importance of A-type lamins for muscle differentiation and/or maintenance (52, 65). The mechanism by which mutations in A-type lamins generate tissue-specific disorders is unknown. However, one model posits that lamin A/C regulates gene expression in differentiated tissue by coordinating the activity of key transcription factors.
A-type lamins localize to peripheral regions underlying the nuclear envelope and also concentrate at perinucleolar foci (7, 31). They are hypothesized to affect gene expression at both peripheral and perinucleolar sites due to interactions with transcription factors involved in differentiation, such as the retinoblastoma protein (pRB) (26, 41, 42, 44, 51, 59, 67). Previously, it has been shown that pRB is tethered to the nuclear substructure through the action of lamin A/C (66). Further, A-type lamins protect pRB, and the related protein p107, from proteasomal degradation (27). pRB regulates cell cycle progression by inhibiting the activity of the transcriptional activator E2F and through establishment of repressive chromatin structures at genes necessary for S-phase entry and progression (2, 4, 16, 22). Entry into and maintenance of cell cycle arrest is essential for cellular differentiation. In addition, pRB is important for muscle and fat cell differentiation (34), as well as cellular senescence (50), suggesting that deregulated pRB activity could play a role in the pathology associated with laminopathies (14, 36, 57, 61, 75, 78, 79).
Overexpression of p16ink4a inhibits cyclin D-dependent kinase activity leading to hypophosphorylated pRB, reduced E2F activity, and arrest in the G1 phase of the cell cycle (60). Cells lacking pRB are refractory to arrest by p16ink4a (8, 33, 38, 43). To determine whether lamin A/C is necessary for pRB-mediated cell cycle regulation, we examined cell cycle arrest in cells lacking lamin A/C. We found that cells lacking A-type lamin function do not arrest in the presence of p16ink4a. p16ink4a-mediated cell cycle arrest was restored by reintroduction of either lamin A or lamin C or increased expression of pRB. Lamin A mutants defective for processing could not restore p16ink4A-mediated arrest or stabilize pRB, suggesting that appropriate modification of lamin A is important for maintaining normal levels of pRB. A mandibuloacral dysplasia (MAD)-associated mutant LMNA allele (R527H) also failed to restore signaling through the p16ink4a/pRB pathway (49, 62). Surprisingly, however, other laminopathy alleles, including another MAD mutant, adequately restored p16ink4a-mediated cell cycle arrest.
Our findings serve as a model for the regulation of transcription factor activity by A-type nuclear lamins. Further, they indicate that A-type lamin-mediated stabilization of pRB is necessary for G1 cell cycle arrest in response to increased CDK inhibitor activity.
| MATERIALS AND METHODS |
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Cell culture and plasmid transfection. Immortalized knockout fibroblast cell lines, as well as litter-matched controls, have been described (12). All cell lines were cultured in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 10 U of penicillin/ml, and 10 µg of streptomycin/ml. Cells grown to 30% confluence were transfected using Lipofectamine Plus (Invitrogen). The following constructs were used: pcDNA3.1 green fluorescent protein (GFP), pcDNA3.1-GFP-p16ink4a, pcDNA3.1-GFP-p21cip1, pcDNA3.1-3xFlag, pcDNA3.1-3xFlag pRB, pcDNA3.1-3xFlag lamin B, pcDNA3.1-HA, and pcDNA3.1-HA-tagged-Ub. To determine the percentage of S-phase cells, cells were grown on coverslips for 36 h, pulsed with bromodeoxyuridine (BrdU) for 6 h, and then fixed with 4% paraformaldehyde and detected by immunofluorescence as previously described (8). The results were recorded from triplicate samples, and 150 cells were scored per sample. Each experiment was repeated three times, and the data presented are a representative sample.
Retroviral infections. The retroviruses pMXIH and pMXIH-lamin A were created as previously described (35). At 36 h after infection, infected cells were selected by culturing in selective media containing 500 µg of hygromycin B/ml for 2 days. The retroviruses pBabe and pBabe-p16ink4a (kindly provided by the Marie Classon Cancer Center, Massachusetts General Hospital) were infected into cells and selected in 6 µg of puromycin/ml for 2 days, while pLXSN and pLXSN-p16ink4a (kindly provided by Denise Galloway, Fred Hutchinson Cancer Research Center) were selected for 5 days using 1 mg of G418/ml. At 24 h before additional transfection, the selective media were removed from the infected cells, and supplemented Dulbecco modified Eagle medium (described above) was added. Infection of retroviral small inhibitory RNA (siRNA) constructs to mouse lamin A was previously described (35). Ecotropic retroviruses of pSuper.retro-Puro (Oligoengine) and pSuper.retro-Puro siLamin A plasmids was prepared as described above. At 24 h after NIH 3T3 cells were infected, cells were selected by using 5 µg of puromycin/ml for 3 days. These cells were used not for experiments more than six passages after selection.
Fluorescence-activated cell sorting (FACS) analysis. At 36 h after transfection, flow cytometry analysis was conducted on the transfected cells in the Rabinovitch Laboratory (Department Pathology, University of Washington). The cells were washed in a solution of 10 µg of DAPI (4',6'-diamidino-2-phenylindole)/ml and 0.1% Nonidet P-40 detergent in a Tris-buffered saline and then triturated with a 26-gauge needle and analyzed by using a Coulter ELITE cytometer (Coulter Corp., Miami, FL) with UV excitation and DAPI emission collected at >450 nm (55). DNA content and cell cycle were analyzed by using the software program WinCycle software (Phoenix Flow Systems, San Diego, CA) as previously described (54). At least 25,000 cells were counted per experiment. The data are presented as histograms in which the cell number is plotted against the DNA content. To determine GFP-positive cells, untransfected cells were used as a control. For each sample, 40,000 GFP-positive cells were collected and analyzed by using Western analysis.
Indirect immunofluorescence. Immunofluorescence was performed on formaldehyde fixed cells except for those stained for lamin A/C (SC-20681), which were methanol fixed as previously described (31). The following antibodies were used: mouse anti-pRB (Pharmingen, [catalog no.] G3-245), rabbit anti-pRB (Neomarkers, Ab-6), rabbit anti-lamin A/C (Santa Cruz, SC-20681), mouse anti-lamin A/C (Santa Cruz, SC-7292), rabbit anti-p16 (Chemicon, Ab3004), and mouse anti-BrdU (Becton Dickinson, 347580). Images were taken by using Zeiss Axiovert 200 (Obserkochen, Germany). Images that compared protein levels were collected using equal exposure times and processed similarly.
Protein analysis and immunoprecipitation. Protein extracts from total cells were harvested in radioimmunoprecipitation assay buffer (0.15 M NaCl, 0.05 mM Tris-HCl (pH 7.2), 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS]). A total of 40 to 50 µg of total protein was separated by denaturing electrophoresis. The following antibodies were used for immunoblotting: mouse anti-pRB (Pharmingen, G3-245), rabbit anti-pRB Ser807/811 (Cell Signaling, 9308), mouse anti-actin (Chemicon MAB1501R), mouse anti-Flag (Sigma, F3165), mouse anti-HA (Convance, MMS-101R), rabbit anti-pan lamin A/C (Cell Signaling, 2032), mouse anti-lamin A/C (Santa Cruz, SC-7292), mouse anti-p21/Cip1/WAF1 (Upstate, 05-345), and rabbit anti-p16 (Chemicon, Ab3004). Immunoprecipitations for 3xFlag constructs were conducted in radioimmunoprecipitation assay buffer using 1 µg of mouse anti-Flag (Sigma, F3165) bound to protein G beads (Roche). Proteasomal studies were conducted by incubating the transfected cells in 25 µM MG132 (Sigma, C2211) and/or 50 µg of cycloheximide/ml (Sigma, R750107). The protein extracts were harvested and immunoblotted as described above. Protein levels were determined by counting pixels using NIH Image (National Institutes of Health).
| RESULTS |
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A recent report has suggested that pRB levels are not reduced in cells lacking lamin A/C and that pRB instead exhibits increased phosphorylation in serum-deprived, G1-arrested cells (69). We show in the present study that two cell lines with reduced A-type lamins (Lmna/ fibroblasts and siLmna-treated NIH 3T3 cells) exhibit increased ubiquitination and degradation of pRB. Nevertheless, we examined the phosphorylation state of pRB in Lmna/ fibroblasts and siLmna-treated NIH 3T3 cells arrested by serum deprivation. We conducted a time course in which Lmna/ and siLmna cells were grown in 0.1% serum for 24 and 48 h. FACS analysis showed that Lmna/ and siLmna require 48 h to become G1-phase arrested, whereas the control cells, Lmna+/+ and siGFP, are largely arrested at 24 h, indicating that cells with reduced lamin A/C have impaired cell cycle withdrawal kinetics (Fig. 3A). Lmna/ myoblasts also exhibit delayed G1-phase cell cycle arrest when induced to differentiate (21). After serum deprivation for 48 h, we examined pRB phosphorylation using a pRB antibody that recognizes pRB phosphorylated at Ser807/Ser811. This same phospho-specific antibody was used in the study by Van Berlo et al. (69). Western analysis indicated that quiescent Lmna/ and Lmna+/+ cells both possessed little to no Ser807/811 phosphorylated pRB (Fig. 3B). Similar findings were observed in serum-deprived siLmna-treated NIH 3T3 cells and in Lmna/ and Lmna+/+ cells arrested by contact inhibition, indicating that G1 arrested cells lacking lamin A/C do not possess higher amounts of phosphorylated pRB (see Fig. S1 in the supplemental material). From our studies, we conclude that pRB is maintained in a highly stable state in the nucleus through the action of lamin A/C.
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We introduced GFP-p16ink4a into Lmna/ and Lmna+/+ fibroblasts, as well as fibroblasts lacking pRB family members, and examined the proliferative state of cells 36 h posttransfection. Cells were labeled for 6 h with BrdU and fixed, and the percentage of cells positive for both GFP and BrdU was determined (Fig. 4A, B, and D). Similar to a previous report using mouse embryo fibroblasts lacking pRB family members (8), GFP-p16ink4a reduced the percentage of S-phase cells in Rb+/+ immortalized fibroblasts dramatically, whereas Rb/ or p107//p130/ fibroblasts had no significant reduction. Importantly, Lmna/ cells were also unresponsive to p16ink4a overexpression, suggesting that lamin A/C is necessary for pRB-dependent cell cycle arrest. To verify that p16ink4a-unresponsiveness was a direct consequence of loss of lamin A/C, we determined the response of siLmna cells to elevated p16ink4a expression (Fig. 4C and D). Consistently, we found that siLmna cells also fail to undergo cell cycle arrest in response to transient p16ink4a expression. Levels of p16ink4a overexpression were comparable in transfected Lmna+/+ and Lmna/ cells and in siLmna cells, relative to loading controls (Fig. 4D).
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To confirm that the loss of lamin A/C compromises cell cycle regulation by pRB and to measure the cell cycle position of p16ink4a-expressing cells, we introduced p16ink4a into Lmna/ cells through retroviral infection. The infected cells were selected with puromycin for 2 days, and the percentage of cells in S phase was determined by quantifying BrdU incorporation, as described above. In all cases, we found that greater than 90% of the puromycin-resistant cells expressed p16ink4a (Fig. 5A). When the percentage of S-phase cells were determined, we found that p16ink4a-infected Lmna/ cells did not have a significant reduction of S-phase cells compared to the vector control, whereas p16ink4a-infected Lmna+/+ cells demonstrated a sharp reduction (Fig. 5B). The expression levels of p16ink4a were similar in Lmna+/+ and Lmna/ cells (Fig. 5C). Flow cytometric analysis was performed to measure more specifically the cell cycle positions of these cells. As expected, we found that p16ink4a confers G1-phase arrest in Lmna+/+ cells but not in cells lacking A-type lamins (Fig. 5D). Similar results were obtained when p16ink4a was transduced into siLmna cells (see Fig. S2 in the supplemental material).
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Reintroduction of pRB induces arrest in cells lacking lamin A/C. We have reported previously that lamin A/C is required for both the stability of pRB and its localization to perinucleolar foci (27). Since proper localization might be required for normal pRB activity, it was not clear whether increased expression of pRB in Lmna/ fibroblasts would be sufficient to restore p16ink4a responsiveness. To test the consequences of increased pRB expression on cell cycle progression in cells with reduced lamin A/C expression, we transfected a variety of cell lines with either GFP alone, GFP and pRB, or GFP-p16ink4a and pRB (Fig. 7A). At 36 h after transfection only 8% of the GFP-pRB-positive Lmna/ cells were also positive for BrdU compared to 33% of the GFP-positive control Lmna/ cells. Similar results were found when GFP-pRB was expressed in littermate Lmna+/+ cells, as well as the siLmna and siGFP cells. This finding is consistent with experiments in NIH 3T3 cells, where enforced pRB expression confers reduced cell proliferation (24, 53, 64), and indicates that increased expression of pRB in cells with reduced lamin A/C expression can still confer G1 arrest. Cotransfection of GFP-p16ink4a and pRB led to a further reduction in the percentage of S-phase cells relative to controls. This finding indicates that the loss of pRB is sufficient to explain the lack of responsiveness of Lmna/ cells to elevated p16ink4a expression.
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Expression of laminopathy-associated and processing-defective lamin A mutants. Given the known roles of pRB in cell cycle control, cell senescence, and muscle and fat cell differentiation, we speculated that reduced pRB activity might underlie some of the pathologies in one or more of the laminopathies (14, 36, 57, 78). To test this, we examined the ability of a number of lamin A mutant alleles, corresponding to missense mutations associated with disease, to restore p16ink4a responsiveness to the Lmna/ fibroblasts. We chose lamin A mutants from mandibuloacral dysplasia (MAD; R527H and A529V), atypical Werner syndrome (AWS; A57P and R133L), Charcot-Marie-Tooth disorder type II (CMT2B1; R298C), Dunnigan-type familial partial lipodystrophy (FPLD; R28W, R62G, and V440M), dilated cardiomyopathy (CMD1A; L85R, N195K, and S573L), limb girdle muscular dystrophy (LGMD; R377H), Emery-Dreifuss muscular dystrophy (EDMD2/3; R453W and R527P), and Hutchinson-Gilford Progeria Syndrome (HGPS; G608G* and R527C). The G608G* allele was engineered to express the lamin A mutant protein corresponding to the allele produced by activation of a cryptic splice site in the G608G HGPS mutant (35). Many if not all of these mutations are linked to a defect in nuclear shape. We suspected that expression of many of these alleles in Lmna/ fibroblasts would be insufficient to restore proper pRB expression and/or p16ink4a-mediated cell cycle arrest. However, with the exception of the R527H MAD allele, expression of all other laminopathy mutants restored responsiveness to p16ink4a (Fig. 8A). Interestingly, the MAD A529V mutant also restored p16ink4a sensitivity, suggesting that loss of pRB function is not disease associated and instead is specific to the mutation of arginine at residue 527. To help us understand the importance of R527, we tested two laminopathy mutants that derive from different mutations of R527 (EDMD2/3 R527P and HPGS R527C). We found that both mutations restored p16ink4a responsiveness, suggesting that the loss of pRB function and levels is specific to the R527H mutation. As a control we expressed 21cip1, observing normal cell cycle arrest in the presence of all laminopathy mutants.
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We also introduced two processing defective alleles of lamin A and determined their affects on p16ink4a-mediated cell cycle arrest. These include C661S, which blocks prenylation, and L647R, which blocks the second proteolytic event leading to mature lamin A. Neither expression of C661S or L647R was sufficient to restore sensitivity to p16ink4a to the Lmna/ cells, indicating that complete processing of lamin A is required to confer pRB stability and function (Fig. 8). The expression and protein levels of the two processing mutants for lamin A/C and pRB were determined in the same manner as for the laminopathy mutants described above (Fig. 8B and C). pRB levels remained low in the presence of both lamin A mutants. Our data correspond to a recent study showing decreased pRB levels in liver cells deficient in lamin A processing (70). It is somewhat surprising that the L647R and G608G* HGPS mutant behaved differently in this assay. G608G activates a cryptic splice site leading to altered LMNA splicing, which ultimately produces a lamin A protein that lacks the second proteolytic cleavage site and is therefore expected to remain stably prenylated (see Discussion) (18).
To better understand the role that lamin A processing has in maintaining pRB stability, we cotransfected GFP-p16ink4a and mutant alleles of human lamin A into fibroblasts possessing endogenous lamin A (Lmna+/+ cells). Cells transfected with two lamin A processing mutants (C661S and L647R) became refractory to p16ink4a, whereas the introduction of wild-type lamin A or MAD R527H did not interfere with p16ink4a responsiveness (Fig. 9A). Thus, processing mutants, but not R527H, act in a dominant fashion to interfere with pRB function. Our data correspond with recent findings suggesting that unprocessed lamin A acts dominantly in laminopathy-based premature aging and that MAD is an autosomal-recessive disorder (37, 40, 48).
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| DISCUSSION |
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Proteasome-dependent degradation of pRB. In mammalian cells, pRB is a highly stable protein, regulated in large part through phosphorylation by cell cycle kinases. The first example of proteasome-dependent pRB degradation was reported for cells expressing the human papillomavirus oncoprotein E7 (6, 28, 46). Recent findings have linked other viral oncoproteins to pRB-mediated degradation including EBNA3C, human T-cell leukemia virus type 1 Tax, and the human cytomegalovirus pp71 protein (29, 30, 32). It is possible that these viral oncoproteins could act at least in part by interfering with pRB access to nuclear lamins or the nuclear matrix. Another recent report found that cells with reduced lamin A/C expression exhibit altered pRB phosphorylation patterns but not reduced stability (69). Previous studies have linked increased pRB phosphorylation to its degradation (25, 39, 73). A-type lamins may be altering phosphorylation of a specific site, leading to pRB instability.
Lamin C rescues pRB levels and function. Prior reports postulated that lamin A is especially important relative to lamin C since lamin A targets lamin C and emerin to the nuclear envelope (56, 71). However, a recent study showed that mice expressing only lamin C were entirely healthy, suggesting that lamin A is a dispensable nuclear component (20). Fong et al. (20) did not investigate the mechanism by which lamin C compensated for the loss of lamin A, but one could hypothesize that lamin A and lamin C possess overlapping functions that are not dependent on each other. Supporting this assertion, we find that reintroduction of either lamin A or lamin C into Lmna/ cells restored p16ink4a sensitivity and pRB protein levels.
LMNA disease mutants. Why missense mutations in LMNA lead to at least 13 different human diseases with several nonoverlapping pathologies remains largely a mystery. Also unresolved is why missense mutations in the same gene can lead to so many sometimes nonoverlapping diseases. We and others have considered the possibility that deregulated pRB function might underlie some of the pathologies associated with LMNA mutation (26, 79). By this reasoning, reintroduction of lamin A disease mutants into Lmna/ cells might not restore pRB protein levels and p16ink4a responsiveness. We chose representative Lamin A mutant alleles from a variety of diseases and transfected them in Lmna/ fibroblasts. All but one of the mutants (see below) restored p16ink4a responsiveness. With regard to these mutants (representing AWS, CMD1A, CMT2B1, EDMD2/3, FPLD, and HGPS), we found no evidence to support the assertion that decreased or altered pRB function is a factor in disease progression.
Surprisingly, only one of the disease-associated mutants, R527H, failed to restore p16ink4a responsiveness. The arginine at residue 527 is particularly interesting since mutation to histidine is associated with MAD, mutation to proline is associated with EDMD2/3, and mutation to cysteine has been linked to HGPS (5, 9, 49). Of note, the patient with MAD was found to be homozygous for R527P, whereas the EDMD2/3 and HGPS patients, like most other affected individuals, carried only one copy of the mutant allele. We tested all three mutants for their ability to restore p16ink4a responsiveness, finding that R527P and R527C complemented lamin A/C deficiency and not R527H. Structural analysis of the C-terminal immunoglobulin region of lamin A suggests that all three mutations should lead to an abrogation of a salt bridge with a glutamate at residue 537 (23). Consequently, the reason for the divergent behavior of these three mutant alleles is unknown. Although the MAD R527H mutant cannot confer pRB stability, we think it unlikely that destabilization of pRB underlies mandibuloacral dysplasia since another MAD mutant (A529V) retains the capacity to stabilize pRB.
Lamin A processing and pRB stability. Mutation of ZMPSTE24 is also known to cause MAD, HGPS, and restrictive dermopathy (another disease linked to LMNA mutation) (1, 40, 47, 49, 68). This zinc metalloprotease is responsible for C-terminal processing of lamin A (13). We have tested the ability of two lamin A mutants that interfere with C-terminal processing (C661S or L647R), finding that neither of them restore pRB stability and cell cycle arrest in response to p16ink4a expression. The C661S mutant blocks processing prior to prenylation, whereas the L647R mutant interferes with the second cleavage event, presumably leading to permanent prenylation. Interestingly, these mutants not only fail restore pRB stability in Lmna/ cells but also interfere with the stabilizing ability of wild-type lamin A/C when expressed in Lmna+/+ cells, indicating that proper maturation of lamin A is required to stabilize pRB and sensitize cells to p16ink4a expression.
The HGPS mutant G608G* results in an aberrant splicing event, removing 50 amino acids in the C terminus of lamin A, including the second processing site. As such, it is expected that this lamin A mutant will remain permanently prenylated. Therefore, we predicted that the HGPS mutant would behave identically to L647R, which removes the second Zmpste24 cleavage site. However, Lmna/ cells expressing L647R had low levels of pRB and were refractory to p16ink4a arrest, whereas Lmna/ cells expressing G608G* exhibited a normal response to p16ink4a overexpression. Thus, the protein that results from the G608G* mutant does not always phenocopy deficient lamin A processing. A recent study supports our finding concerning the L647R mutant since liver cells from a Zmpste24/ mouse possessed decreased pRB levels (70). Although regulation of pRB may be unrelated to HGPS, this finding, along with the fact that other lamin A mutants linked to HGPS have no obvious link to C-terminal processing, should serve as a cautionary note that the pathologies associated with lamin mutations causing HGPS may not be entirely explained by lamin A processing defects.
Summary. A-type lamins are thought to regulate the activity of a number of transcription factors, although the precise mechanism(s) by which this intermediate filament protein regulates transcription remains unknown. We show that lamin A/C controls the stability of pRB and is required for its function in cell cycle control. Although lamin A/C is reported to interact with pRB, the relevance of this interaction to the maintenance of pRB stability remains to be determined. We speculate that lamin A/C binds to pRB and either promotes proper localization of pRB, interferes directly with pRB ubiquitination, or both. Association with the nuclear substructure is a characteristic of many transcription factors, including pRB, and continued studies of lamin A and pRB will likely shed light on the relationship between nuclear structure and proper transcriptional control.
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| ACKNOWLEDGMENTS |
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R.T.N. is supported by NIH training grant AG013280. B.A.K. is supported in part by PHS NRSA T32 GM07270 from the NIGMS. This research was supported by National Institutes of Health grant R01AG024287 to B.K.K. B.K.K. is a Searle Scholar.
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Supplemental material for this article may be found at http://mcb.asm.org/. ![]()
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