Vol. 59, No. 2
INFECTION AND IMMUNITY, Feb. 1991, p. 471-477
0019-9567/91/020471-07$02.00/0
Environmental Conditions Which Influence Mucoid Conversion in Pseudomonas aeruginosa PAO1 JAMES M. TERRY, SOPHIA E. PINA, AND STEPHEN J. MATTINGLY* Department of Microbiology, University of Texas Health Science Center at San Antonio,
San Antonio, Texas 78284 Received 24 August 1990/Accepted 8 November 1990
Growth and conversion to the mucoid phenotype by nonmucoid Pseudomonas aeruginosa PAO1 was studied in a chemostat system under conditions designed to reflect those likely to be present during chronic infection in the lung in cystic fibrosis patients. Mucoid variants were consistently isolated during continuous culture in the presence of 0.3 M NaCl or 5 or 10% glycerol. Mucoid subpopulations were also detected under conditions of carbon, nitrogen, or phosphate limitation. During carbon or nitrogen limitation, mucoid conversion was dependent upon the choice of substrate. Phosphate-limited cultures exhibited an inverse relationship between culture growth rate and number of mucoid organisms detected. Mucoid variants were not detected when dilution rates (D) exceeded 0.173 h-'. Conversely, at a D of 0.044 h-1, 40% of the population expressed the mucoid phenotype. Phosphorylcholine, a product of phosphojipase C activity on the major lung surfactant phosphatidylcholine, was also used as a growth substrate in nutrient limitation studies. Under all conditions, growth of PAO1 supplied with phosphorylcholine resulted in isolation of mucoid variants, indicating that the lung may provide at least one nutrient source conducive to mucoid conversion. Continuous culture also resulted in detection of a phage associated with strain PAO1. High titers of phage were present under all conditions, including those which yielded no mucoid organisms, suggesting that environmental conditions rather than the phage regulated the appearance of mucoid variants.
which might have relevance within the context of chronic lung infections, such as slow growth rate, limitation of nutrients, utilization for growth of components found in lecithin, and high medium osmolarity, resulted in the appearance of a persistent mucoid subpopulation. The emergence of mucoid organisms usually coincided with the appearance of bacteriophage activity in these cultures. However, phage activity was detected under all growth conditions, while mucoid organisms were only detected when certain environmental or nutritional conditions existed. The possible relevance of these results to the conversion of P. aeruginosa to the mucoid phenotype in chronic lung infections is discussed.
Pseudomonas aeruginosa is responsible for much of the morbidity and mortality associated with chronic pulmonary infections in cystic fibrosis (CF) patients (10, 15, 24). With prolonged chronic infection, P. aeruginosa undergoes alteration from the classical nonmucoid form to an atypical mucoid form, which correlates with the production of the exopolysaccharide alginic acid (6). In the context of chronic lung infection, alginate is an important virulence determinant. The appearance of alginate-producing P. aeruginosa strains is usually associated with a poor patient prognosis (10). Alginate has been implicated in adherence to tracheal epithelium (21, 28) and has been associated with antiphagocytic function (29). While the relationship between mucoid P. aeruginosa and CF is well documented, the environmental factors of the CF lung responsible for conversion of nonmucoid P. aeruginosa to the alginate-producing mucoid form have yet to be defined. A chemostat model for the study of this organism under chronic diseaselike growth conditions (phosphate and nitrogen limitation) which would reflect the likely physiological status of organisms in the lungs of CF patients has recently been reported (18). In this study, phosphate-limited cultures of mucoid P. aeruginosa were capable of utilizing phosphorylcholine (PC), a product of phospholipase C activity on phosphatidylcholine (a major component of lung surfactant), as a sole source of phosphate for growth (18). In an effort to understand the environmental factors responsible for the conversion of nonmucoid P. aeruginosa to the mucoid phenotype during chronic lung infection in CF patients, studies have been extended to investigation of the growth of nonmucoid P. aeruginosa in a chemostat system. In this report, prolonged continuous culture of nonmucoid P. aeruginosa PAO1 under several environmental conditions *
MATERIALS AND METHODS
Maintenance of bacteria. P. aeruginosa PAO1 was obtained from B. Iglewski, Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, N.Y. All stocks were maintained at -80°C in 10% skim milk and subcultured on mucoid maintenance agar (MMA) plates (MacConkey agar base modified by the addition of 50 g of glycerol per liter) (7). Culture media. A modification of the chemically defined alginate-promoting (AP) medium of Mian et al. (25), which has been described previously (17, 18), was the primary growth medium used for the chemostat studies. The medium contained 100 mM monosodium glutamate, 100 mM sodium gluconate, 7.5 mM NaH2PO4, 16.3 mM K2HPO4, and 10 mM MgSO4 7H20. The pH was adjusted to 7.0 with 2.5 N NaOH. For growth under various environmental conditions, further modifications of this medium were necessary. These modifications are summarized in Table 1. Continuous-culture conditions. Continuous culture was performed in a New Brunswick Bioflo bench-top chemostat (model C30; New Brunswick Scientific Co., Inc., Edison,
Corresponding author. 471
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TABLE 1. Growth media used for continuous culture of PAO1 Medium components AP' plus osmotic agent (0.3 M NaCl, 0.4 M sucrose, or 5 or 10% glycerol) High osmolarity ....................... AP plus monosodium glutamate reduced to 1 mM or replaced by 1 mM Nitrogen limitation (N medium) ....................... NaNO3 Carbon limitation (C medium) ....................... 2 mM K2HPO4, 18 mM NH4Cl, 10 mM MOPS,b 1 mM MgSO4 7H20, 2 mM carbon source, pH adjusted to 7.0 AP with NaH2PO4 and K2HPO4 replaced by 0.05 mM K2HPO4, 10 mM Phosphate limitation (P medium) ....................... MOPS added as a buffer, pH adjusted to 7.0 PC P medium with phosphate replaced by 10 mM PC chloride Phosphate source ....................... 2 mM K2HP04, 10 mM MgSO4 7H20, 100 mM sodium gluconate, 10 mM Nitrogen source ....................... MOPS, 1 mM PC chloride, pH adjusted to 7.8 Carbon source ....................... C medium with 2 mM PC chloride as sole carbon source Sole source of C, N, and P04 ..................... 10 mM PC chloride, 10 mM MgSO4 7H20, 10 mM MOPS, pH 7.0
Culture conditions
aAP, Alginate-promoting medium. MOPS, 3-[N-Morpholinolpropanesulfonic acid.
b
N.J.) with either a 350-ml or a 1.2-liter working volume. Culture conditions were as described previously (18) with the following modifications. When anaerobic conditions were required, nitrogen gas was supplied to the cultures at a rate of 0.5 liter min-', and sodium nitrate was added to the medium at a final concentration of 2% to serve as an alternative electron acceptor (31, 34). For continuous-culture studies which examined growth under conditions of high medium osmolarity, the addition of medium from the reservoir was initiated when the batch cultures reached the mid-exponential phase. All cultures were maintained at a specified dilution rate for several days. Growth and colony morphology. Samples were obtained from the culture vessel at various time points during growth of strain PA01. Appropriate dilutions were made in AP medium, and 0.1-ml aliquots of these samples were plated onto MMA plates for determination of growth based upon colony-forming units (CFU) per milliliter. The phenotypic expression of the culture was determined at the same time by noting the number of each colony type on MMA plates. Production of alginate by representative mucoid colonies was verified by isolation and purification of alginate by previously described methods (18), and the stability of mucoid colonies was determined by a minimum of three passages on MMA. Bacteriophage determinations. Plaque assays were performed with filtrates made from samples taken directly from chemostat cultures. Filtrates were diluted in phage suspension medium (SM) containing 100 mM NaCl, 10 mM MgSO4 .7H20, 0.1% gelatin, and 50 mM Tris, pH 7.5 (20). Aliquots (0.1 ml) were spread over 0.5% soft agar lawns of PA01. Following overnight incubation at 37°C, plaques were counted and phage activity was determined in plaque-forming units (PFU) per milliliter. Samples which contained phage activity were pooled and stored at 4°C for further use in phage lysate preparations. Phage stocks were made from plate lysates prepared from plaque-purified phage samples. Stocks were stored frozen in SM with 15% glycerol at -80'C or in SM at 4°C until used for phage sensitivity and adsorption assays. Phage sensitivity and adsorption assays. Nonmucoid colonies from plated samples of chemostat cultures taken at different times during the course of these cultures were screened for phage sensitivity. These colonies were subcultured first onto MMA plates. Only those colonies which did not spontaneously lyse after 48 h of incubation at 370C were tested for phage sensitivity. Colonies were resuspended in
AP medium and grown overnight in 50-ml cultures at 37°C with constant shaking. A 0.1-ml volume of each culture was spread onto MMA plates, which were allowed to dry for 60 min. Aliquots (50 ,u) of phage preparations were spotted onto these plates. After overnight incubation at 37°C, plates were checked for zones of clearing where phage had been spotted. Mucoid PAO1 variants isolated from cultures were treated in the same manner except that lawns were made in 0.5% soft agar in order to reduce the amount of exopolysaccharide production, which often obscured signs of phage activity. Plates from the original PA01 stock were also treated with phage preparations as a positive control. Mucoid variants were also tested for sensitivity by infection of soft-agar lawns with diluted phage samples and comparison with the number of plaques formed in nonmucoid PAO1 lawns infected with identical phage preparations. Mucoid variants were also compared with nonmpcoid PA01 in adsorption assays for their ability to adsorb phage. Triplicate samples of 108 to 109 cells from overnight cultures were mixed with an equal volume of SM containing 104 to 105 PFU of phage in microfuge tubes. After a 40-min incubation at 37°C, the mixtures were centrifuged at 1,100 x g for 15 min to get rid of cells, and supernatant fluids were subjected to sterile filtration. Samples were thep diluted in SM, and 0.1-ml volumes of each were spread onto soft-agar lawns of PAO1. Plates were incubated overnight, and plaques were counted. Phage adsorption was calculated from the reduction in the phage titer of samples compared with the titers of identical phage preparations which had not been exposed to cells. Statistically significant differences were assessed by a two-tailed Student's t test for independent means (P - 0.05 was considered not significant). RESULTS Osmotic stress. Growth of P. aeruginosa PAQ1 under conditions of high medium osmolarity was accomplished by continuous culture of organisms in AP medium with either 0.3 M NaCl or 0.4 M sucrose as osmotically active agents. Additionally, growth in AP medium with either 5 or 10% (vol/vol) glycerol, an osmotically neutral agent, was investigated. The results of these experiments were compared with those in continuous culture in AP medium alone (Table 2). Cultures were maintained for several days; however, it was difficult to achieve a steady state of growth as defined by constant CFUJ per milliliter on MMA plates for more than 48 to 60 h. Under all growth conditions, within the first 30 h of
MUCOID CONVERSION OF P. AERUGINOSA PAO1
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TABLE 2. Effect of increased osmolarity on mucoid conversion of P. aeruginosa PAO1 Osmotic agent
Concn
NaCI Sucrose Glycerol
0.3 M 0.4 M 5%
10% Nonec
D
% Mucoid
(h-1)
coloniesa
0.094 0.104 0.097
2.9
0.132 0.139
O.Ob
TABLE 3. Mucoid conversion of strain PAO1 during nitrogen or carbon limitation Growth conditiona
Nitrogen limitation Glutamate
3.5
5.6
Nitrate
O.Ob
a Percentage of colonies which expressed the mucoid phenotype at the end of the growth experiment. b No mucoid colonies detected during the growth experiment. I AP medium alone.
culture, there was a significant decrease in culture viability coincident with the appearance of two new and unusual colony morphotypes on MMA plates. One colony was of normal size but appeared to be undergoing lysis and completely disintegrated over time. The second colony type was small and slow growing and began to lyse within 48 h. Resuspension of these colonies in AP medium and inoculation of filtered supernatant fluids onto soft-agar lawns of PAO1 yielded bacteriophage plaques after overnight incubation at 37°C. Thus, it appeared that during the course of continuous culture of P. aeruginosa PAO1, a bacteriophage was produced. Following the appearance of the lysing colonies and evident bacteriophage activity, mucoid colonies appeared on MMA plates inoculated from cultures of PAO1 grown in the presence of 0.3 M NaCl, 5% glycerol, or 10% glycerol (Table 2). The percentage of the viable culture population which expressed the mucoid phenotype at the end of each growth experiment was 2.9, 3.5, and 5.6% for growth in 0.3 M NaCl, 5% glycerol, and 10% glycerol, respectively. No mucoid variants were detected when 0.4 M sucrose was used as the osmotic agent or when strain PAO1 was grown in AP medium alone (Table 2). Nitrogen limitation. Glutamate, a rapidly assimilated nitrogen source, and nitrate, a poorer source of nitrogen, were chosen for the investigation of the effect of nitrogen limitation on mucoid conversion of P. aeruginosa PAO1. Growth in N medium with 1 mM glutamate as the nitrogen source yielded no mucoid organisms, even at very slow growth rates. Conversely, at slower growth rates in cultures supplied with nitrate, a subpopulation of mucoid variants was detected (Table 3), with similar cultures yielding from 0.1 to 0.4% of viable organisms expressing the mucoid phenotype on MMA plates. High bacteriophage titers were observed in all cultures, but only cultures grown with nitrate as the sole nitrogen source produced mucoid organisms (Fig. 1). Carbon limitation. Four carbon sources, acetate, glycerol, succinate, and gluconate, were chosen for carbon limitation experiments. Cultures were maintained at low dilution rates (D), which ranged from 0.038 to 0.058 h-'. Again, high titers of bacteriophage were detected during each experiment. Gluconate-limited growth failed to yield mucoid PAO1 variants. In contrast, when the growth-limiting carbon sources were composed of fewer carbons, mucoid organisms appeared consistently. For acetate, glycerol, and succinate, the level of mucoid organisms was 2.8, 1.3, and 4.4%, respectively (Table 3). Figure 2 illustrates a comparison between growth in gluconate- and growth in succinatesupplemented C medium (Fig. 2A and B, respectively). Interestingly, 3% of the succinate-grown organisms were
473
Carbon limitation Acetate Glycerol Succinate Gluconate
D
% Mucoid
(h-')
coloniesb
0.032 0.353 0.046 0.045
0.OC 0.Oc
0.058 0.046 0.046 0.038
2.8 1.3 4.4
0.4 0.1
0.0O
a For nitrogen limitation, 1 mM nitrogen source; for carbon limitation, 2 mM carbon source. b Percentage of colonies which were mucoid at the end of the growth
experiment. ' No mucoid colonies detected during culture period.
mucoid almost 24 h before any phage activity was detected (Fig. 2B). Effect of growth rate on phenotypic expression in phosphate-limited cultures. The effect of decreased growth rates on nonmucoid organisms, as might be found during prolonged chronic P. aeruginosa lung infection, was examined in phosphate-limited cultures of strain PAO1. Dilution rates (D) ranged from 0.035 to 0.347 h-1. Mucoid variants were detected only when D was below 0.347 h-1. The percentage of mucoid organisms in cultures ranged from 0.8% at a D of 0.173 h-1 to 40% at a D of 0.044 h-1 (Table 4). High titers of bacteriophage were detected under all conditions. However, these results were independent of bacteriophage activity, as cultures grown at a relatively high D of 0.347 h-1 (Table 4) or grown anaerobically yielded no mucoid organisms (data not shown). Phenotypic expression in cultures supplied with PC as the limited nutrient source. Continuous culture of strain PAO1 with a growth-limiting amount of substrate likely to be found in the lungs was investigated. PC, used previously in phosphate limitation studies with mucoid organisms (18), was used as the growth-limiting phosphate, nitrogen, or carbon source. Also, this compound was evaluated as the sole source of carbon, nitrogen, and phosphate. Growth of phosphate-limited organisms supplied with 10 mM PC as the phosphate source resulted in expression of the mucoid phenotype by 0.35% of the viable organisms by the end of the culture period (Table 5). Mucoid colonies were evident by day 2 of culture, following the appearance of bacteriophage activity. When PC was supplied as the limiting nitrogen source, 0.49% of the population was mucoid by the end of the culture period (Table 5). Mucoid colonies were detected almost 24 h before phage activity. Cultures of strain PAO1 supplied with PC as the growth-limiting carbon source or as the sole source of carbon, nitrogen, and phosphate yielded 0.1 and 0.88% mucoid organisms (Table 5). Mucoid colonies were observed more than 48 h prior to detectable phage activity in the latter culture. Significance of bacteriophage in PAO1 continuous cultures. Although high levels of bacteriophage were produced under all growth conditions in continuous cultures of nonmucoid PAO1, mucoid variants were observed only when cultures were grown at a relatively low D and only in the presence of high salt or high glycerol (0.3 M NaCl or 5 or 10% glycerol, Table 2) or under limitation for specific nutrients (Tables 3 to
INFECT. IMMUN.
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474
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10
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0.
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FIG. 1. Growth and phenotypic expression of P. aeruginosa PA01 during nitrogen-limited growth in N medium with (A) glutamate and (B) nitrate as the nitrogen source. Symbols: 0, viable organisms; 0, phage activity. The percentage of the culture population which expressed the mucoid phenotype is shown by solid bars in the graph below each growth curve.
5). Additionally, screening of colonies isolated from chemostat cultures revealed that the culture population, both mucoid variants and nonmucoid PAO1, remained sensitive to this phage. Isolation of phage-resistant variants of PAO1 was successful only early in cultures, during the initial burst of phage activity when the cultures went into decline. Thereafter, this subpopulation of the cultures became undetectable as the cultures recovered toward more stable growth. Phage-resistant variants grown under phosphatelimited conditions which yielded 40% mucoid organisms from the original PAO1 culture yielded much lower levels of mucoid organisms. However, these variants have not been characterized further with respect to differences from PAO1 other than phage resistance. Mucoid PAO1 variants were tested for phage sensitivity, and all proved to be sensitive even though phage adsorption assays showed significantly lower adsorption of this phage to mucoid variants than to nonmucoid PAO1 (data not shown). Finally, phage production in batch cultures was investigated. Phage could only be detected in cultures inoculated from MMA plate cultures which were 1 week or more in age. These cultures did not yield mucoid variants. DISCUSSION
Mucoid variants of P. aeruginosa have been recovered from chronic lung infections associated with CF and chronic obstructive lung disease as well as from chronic urinary tract infections (13). More recently, mucoid variants of P. aeruginosa PAO were isolated from the lungs of chronically infected rats (32). Speert et al. (30) have reported isolation of mucoid organisms from prolonged static cultures in acetamide broth. Therefore, conversion to mucoidy is not unique to the CF lung but may take place in response to common
environmental factors encountered during chronic infection or in vitro under adverse growth conditions. The environmental triggers for mucoid conversion are not yet understood. The purpose of this investigation was to determine the environmental conditions conducive to mucoid conversion during continuous culture of a nonmucoid strain. Experiments were designed to reflect conditions which might be present during chronic infection of the CF respiratory tract. One environmental factor relevant to CF is osmotic stress. Exocrine secretions in CF contain elevated sodium, chloride, and calcium ion levels (16), which would result in increased osmolarity of bronchial secretions. Growth of P. aeruginosa PAO1 in 0.3 M NaCl resulted in conversion of 2.9% of the organisms to mucoidy. Enhanced transcription of the biosynthetic algD gene has been reported under similar conditions (2). Ionic strength may be more important than the osmotic component, however, since no mucoid variants were detected during growth in 0.4 M sucrose. Similar differential effects have been noted for amino acid uptake in osmotically stressed Escherichia coli (9). Growth in 5 or 10% glycerol, an osmotically neutral agent, also resulted in isolation of mucoid variants. Excess glycerol can cause formation of toxic metabolites (8, 19). Perhaps these mucoid variants were derived from a subpopulation of organisms more acutely sensitive to this metabolic stress. A second environmental factor would be nutritional limitation. Chronic persistence of P. aeruginosa on mucosal surfaces in the lungs without extensive tissue invasion would require prolonged survival in an environment limited for nutrients. Limitation of nutrients would greatly affect the physiological status of P. aeruginosa, particularly intracellular energy metabolism. Growth rates would have to decrease and organisms would need to become more efficient at utilizing limited nutrients. The results of the phosphate
MUCOID CONVERSION OF P. AERUGINOSA PA01
VOL. 59, 1991
A
475
B
-u
.1-In
.40 C
CD
E
-'
0
0
.c
3
C 0 c
10
0
N..
75
3
In
0
c
2.0.
o
D
0 ,
5k0-0
u
1 2 3 4 5 6 7 8 9 10 11 121314
Time (Days) as
FIG. 2. Phenotypic expression of P. aeruginosa PAO1 during carbon-limited growth in C medium with (A) gluconate and (B) succinate the carbon source. Symbols are the same as in Fig. 1.
limitation experiments are consistent with this idea. At the highest dilution rate (0.347 h-1), mucoid variants were not seen. At very low dilution rates (0.035 to 0.044 h-1), cultures had access to phosphate concentrations well below the 1 mM level known to induce genes responsive to phosphate deprivation in E. coli (23). Phosphate limitation has a direct impact on intracellular energy pool levels, which is probably the reason for the relationship observed between growth rate and the percentage of mucoid organisms in these cultures. The results of the carbon and nitrogen limitation experiments also indicate a relationship between energy metabolism and mucoid conversion. Carbon-limited organisms grown on substrates other than gluconate would experience a greater energy drain due to the increased demand for more tricarboxylic acid cycle intermediates for biosynthetic reactions. No mucoid organisms were isolated when gluconate was used as the carbon TABLE 4. Effect of growth rate on mucoidy in phosphatelimiteda cultures of PAO1 D (h-1)
% Mucoid coloniesb 38.6 0.035 ..................................... 40.0 0.044 ..................................... 2.0 0.069 ..................................... 1.0 0.116 ..................................... 0.8 0.173 ..................................... 0.0 0.347 ..................................... a 0.05 mM K2HPO4. b Percentage of colonies which were mucoid at the end of the growth experiment. c No mucoid colonies detected during the culture period.
source. Likewise, nitrate assimilation requires a larger expenditure of potential energy than glutamate assimilation, in the form of reducing equivalents (27). Mucoid organisms were only detected when the limited nitrogen source was nitrate. Changes which occur during nutritional limitation or starvation have been studied in various organisms (14, 23). Exopolysaccharide synthesis has also been demonstrated during starvation stress (33). The results from our studies with P. aeruginosa PAO1 suggest that mucoid variants may first arise during chronic infection in response to nutrient limitation. Once these variants appear, alginate would afford them protection from host defense mechanisms such as phagocytosis (29), allowing them to persist. The lung may offer a variety of alternative nutrient sources which are liberated by the action of P. aeruginosa exoenzymes. PC is liberated from the major lung surfactant phospholipid phosphatidylcholine by the action of P. aerug-
TABLE 5. Mucoid conversion in PAO1 cultures supplied with PC as an alternative nutrient source Limited nutrienta
Carbon
Nitrogen Phosphate C, N, and P04
D
% Mucoid
(h-1)
coloniesb
0.046 0.044 0.111 0.059
0.10 0.49 0.35 0.88
a For carbon, nitrogen, phosphate, and combined (C, N, and P04) limitation, PC was used at 2, 1, 10, and 10 mM, respectively. b Percentage of colonies which expressed the mucoid phenotype at the end of the culture period.
476
TERRY ET AL.
inosa phospholipase C (1). Phosphate-limited cultures of mucoid organisms have been shown to utilize this substance for alginate production and as a source of phosphate for growth (18). PC offers P. aeruginosa the phosphate, carbon, and nitrogen needed for growth. Mucoid variants of PAO1 were isolated from carbon-, nitrogen-, and phosphate-limited cultures supplied with PC as the sole source of these nutrients. Thus, the environment of the lung offers a potential growth substrate for P. aeruginosa, and utilization of this substrate for growth can yield mucoid variants. While nutrient limitation experiments resulted in isolation of mucoid variants of PAO1, all growth conditions also resulted in production of a bacteriophage associated with this organism. Whether the bacteriophage which was detected was a clear-plaque mutant of a resident lysogenic phage or was a phage existing in a pseudolysogenic state is not yet known. However, the phage was able to infect and lyse strain PAO1. Although phage-mediated mucoid conversion has been reported (22, 26), our results suggest that environmental conditions regulated the appearance of mucoid variants. All mucoid variants tested proved to be sensitive to this phage. Nonmucoid phage-resistant variants of PAO1 were only detected in chemostat cultures during the period of the initial burst of phage activity, when these cultures went into decline. During recovery of these cultures and the return to more stable growth, this resistant subpopulation became undetectable. If the phage was a significant selective agent, selection of resistant organisms, which would predominate over sensitive subpopulations, would be expected. At no time was this the case. Clearly, the results of these studies with P. aeruginosa PAO1 indicate that many environmental factors can play a role in mucoid conversion. The conversion process is not an all-or-nothing response by organisms to environmental stresses but seems to be regulated by the degree to which subpopulations of cells are sensitive to those stresses. There are several widely dispersed genes involved in regulation and synthesis of alginate (3, 5, 11, 12). In nature or in chronic infection, P. aeruginosa must adapt to several environmental stresses. The regulatory algR gene may be part of a sensory transduction circuit responsive to one or more environmental signals which trigger activation of algD and other alg biosynthetic genes (4). The roles of other regulatory genes are not yet clear. Isolation of mucoid variants by continuous-culture methods which allow control of environmental conditions without the use of mutagenic agents is a strategy which might be useful in this pursuit. Analysis of mucoid variants obtained in this way will help in the determination of the genes involved in the complex regulation of alginate synthesis. ACKNOWLEDGMENTS We thank John J. Maurer for his valuable technical assistance and John Govan and Vojo Deretic for their many helpful discussions. This research was supported by grant Z070 from the Cystic Fibrosis Foundation. J.M.T. was supported, in part, by Public Health Service training grant T32 AI-07271 from the National
Institutes of Health.
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INFECT. IMMUN. 3. Darzins, A., and A. M. Chakrabarty. 1984. Cloning of genes controlling alginate biosynthesis from a mucoid cystic fibrosis isolate of Pseudomonas aeruginosa. J. Bacteriol. 159:9-18. 4. Deretic, V., R. Dikshit, W. M. Konyecsni, A. M. Chakrabarty, and T. K. Misra. 1989. The algR gene, which regulates mucoidy in Pseudomonas aeruginosa, belongs to a class of environmentally responsive genes. J. Bacteriol. 171:1278-1283. 5. Deretic, V., J. F. Gill, and A. M. Chakrabarty. 1987. Gene algD coding for GDP mannose dehydrogenase is transcriptionally activated in mucoid Pseudomonas aeruginosa. J. Bacteriol. 169:351-358. 6. Doggett, R. G., G. M. Harrison, R. N. Stilwell, and E. S. Wallis. 1966. An atypical Pseudomonas aeruginosa associated with cystic fibrosis of the pancreas. J. Pediatr. 68:215-221. 7. Evans, L. R., and A. Linker. 1973. Production and characterization of the slime polysaccharide of Pseudomonas aeruginosa. J. Bacteriol. 116:915-924. 8. Freedburg, W. B., W. S. Kistler, and E. C. C. Lin. 1971. Lethal synthesis of methylglyoxal by Escherichia coli during unregulated glycerol metabolism. J. Bacteriol. 108:137-144. 9. Gehring, K., M. Hofnung, and H. Nikaido. 1990. Stimulation of glutamine transport by osmotic stress in Escherichia coli K-12. J. Bacteriol. 172:4741-4743. 10. George, R. H. 1987. Pseudomonas infection in cystic fibrosis. Arch. Dis. Child. 62:431-439. 11. Goldberg, J. B., and D. E. Ohman. 1984. Cloning and expression in Pseudomonas aeruginosa of a gene involved in the production of alginate. J. Bacteriol. 158:1115-1121. 12. Goldberg, J. B., and D. E. Ohman. 1987. Construction and characterization of Pseudomonas aeruginosa algB mutants: role of algB in high-level production of alginate. J. Bacteriol. 169:1593-1602. 13. Govan, J. R. W. 1988. Alginate biosynthesis and other unusual characteristics associated with the pathogenesis of Pseudomonas aeruginosa in cystic fibrosis, p. 67-96. In E. Griffiths, W. Donachie, and J. Stephen (ed.), Bacterial infections of respiratory and gastrointestinal mucosae. IRL Press, Oxford. 14. Harder, W., and L. Dijkuizen. 1983. Physiological responses to nutrient limitation. Annu. Rev. Microbiol. 37:1-23. 15. Hoiby, N. 1974. Epidemiological investigations of the respiratory tract bacteriology in patients with cystic fibrosis. Acta Pathol. Microbiol. Scand. Sect. B 82:541-550. 16. Kilbourne, J. P. 1978. Bacterial content and ionic composition of sputum in cystic fibrosis. Lancet i:334. 17. Krieg, D. P., J. A. Bass, and S. J. Mattingly. 1986. Aeration selects for mucoid phenotype of Pseudomonas aeruginosa. J. Clin. Microbiol. 24:986-990. 18. Krieg, D. P., J. A. Bass, and S. J. Mattingly. 1988. Phosphorylcholine stimulates capsule formation of phosphate-limited mucoid Pseudomonas aeruginosa. Infect. Immun. 56:864-873. 19. Lin, E. C. C. 1976. Glycerol dissimilation and its regulation in bacteria. Annu. Rev. Microbiol. 30:535-578. 20. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 21. Marcus, H., and N. R. Baker. 1985. Quantitation of adherence of mucoid and nonmucoid Pseudomonas aeruginosa to hamster tracheal epithelium. Infect. Immun. 47:723-729. 22. Martin, D. R. 1973. Mucoid variation in Pseudomonas aeruginosa induced by the action of phage. J. Med. Microbiol. 6:111-118. 23. Matin, A., E. A. Auger, P. H. Blum, and J. E. Schultz. 1989. Genetic basis of starvation survival in nondifferentiating bacteria. Annu. Rev. Microbiol. 43:293-316. 24. Mearns, M. B., G. H. Hunt, and R. Rushworth. 1972. Bacterial flora of the respiratory tract in patients with cystic fibrosis, 1950-1971. Arch Dis. Child. 62:431-439. 25. Mian, F. A., T. R. Jarman, and R. C. Righelato. 1978. Biosynthesis of exopolysaccharide by Pseudomonas aeruginosa. J. Bacteriol. 134:418-422. 26. Miller, R. V., and V. J. R. Rubero. 1984. Mucoid conversion by phages of Pseudomonas aeruginosa strains from patients with cystic fibrosis. J. Clin. Microbiol. 19:717-719.
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27. Mortenson, L. E. 1962. Inorganic nitrogen assimilation and ammonia incorporation, p. 119-166. In I. C. Gunsalus and R. Y. Stanier (ed.), The bacteria, vol. III. Academic Press, New York. 28. Ramphal, R., and G. B. Pier. 1985. Role of Pseudomonas aeruginosa mucoid exopolysaccharide in adherence to tracheal cells. Infect. Immun. 47:1-4. 29. Schwarzmann, S., and J. R. Boring. 1971. Antiphagocytic effect of slime from a mucoid strain of Pseudomonas aeruginosa. Infect. Immun. 3:762-767. 30. Speert, D. P., S. W. Farmer, M. E. Campbell, J. M. Musser, R. K. Selander, and S. Kuo. 1990. Conversion of Pseudomonas aeruginosa to the phenotype characteristic of strains from patients with cystic fibrosis. J. Clin. Microbiol. 28:188-194.
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31. Van Hartingsvelt, J., M. G. Marinus, and A. H. Stouthamer. 1971. Mutants of Pseudomonas aeruginosa blocked in nitrate or nitrite dissimilation. Genetics 67:469-482. 32. Woods, D. E., P. A. Sokol, L. E. Bryan, D. G. Storey, S. J. Mattingly, H. J. Vogel, and H. Ceri. J. Infect. Dis., in press. 33. Wrangstadh, M., P. L. Conway, and S. Kjelleberg. 1989. The role of an extracellular polysaccharide produced by the marine Pseudomonas sp. S9 in cellular detachment during starvation. Can. J. Microbiol. 35:309-312. 34. Yamanaka, T., A. Ota, and K. 0. Okanuki. 1961. A nitrite reducing system reconstituted with purified cytochrome components of Pseudomonas aeruginosa. Biochim. Biophys. Acta 53:294-308.
INFECTION AND IMMUNITY, Feb. 1991, p. 471-477
0019-9567/91/020471-07$02.00/0
Environmental Conditions Which Influence Mucoid Conversion in Pseudomonas aeruginosa PAO1 JAMES M. TERRY, SOPHIA E. PINA, AND STEPHEN J. MATTINGLY* Department of Microbiology, University of Texas Health Science Center at San Antonio,
San Antonio, Texas 78284 Received 24 August 1990/Accepted 8 November 1990
Growth and conversion to the mucoid phenotype by nonmucoid Pseudomonas aeruginosa PAO1 was studied in a chemostat system under conditions designed to reflect those likely to be present during chronic infection in the lung in cystic fibrosis patients. Mucoid variants were consistently isolated during continuous culture in the presence of 0.3 M NaCl or 5 or 10% glycerol. Mucoid subpopulations were also detected under conditions of carbon, nitrogen, or phosphate limitation. During carbon or nitrogen limitation, mucoid conversion was dependent upon the choice of substrate. Phosphate-limited cultures exhibited an inverse relationship between culture growth rate and number of mucoid organisms detected. Mucoid variants were not detected when dilution rates (D) exceeded 0.173 h-'. Conversely, at a D of 0.044 h-1, 40% of the population expressed the mucoid phenotype. Phosphorylcholine, a product of phosphojipase C activity on the major lung surfactant phosphatidylcholine, was also used as a growth substrate in nutrient limitation studies. Under all conditions, growth of PAO1 supplied with phosphorylcholine resulted in isolation of mucoid variants, indicating that the lung may provide at least one nutrient source conducive to mucoid conversion. Continuous culture also resulted in detection of a phage associated with strain PAO1. High titers of phage were present under all conditions, including those which yielded no mucoid organisms, suggesting that environmental conditions rather than the phage regulated the appearance of mucoid variants.
which might have relevance within the context of chronic lung infections, such as slow growth rate, limitation of nutrients, utilization for growth of components found in lecithin, and high medium osmolarity, resulted in the appearance of a persistent mucoid subpopulation. The emergence of mucoid organisms usually coincided with the appearance of bacteriophage activity in these cultures. However, phage activity was detected under all growth conditions, while mucoid organisms were only detected when certain environmental or nutritional conditions existed. The possible relevance of these results to the conversion of P. aeruginosa to the mucoid phenotype in chronic lung infections is discussed.
Pseudomonas aeruginosa is responsible for much of the morbidity and mortality associated with chronic pulmonary infections in cystic fibrosis (CF) patients (10, 15, 24). With prolonged chronic infection, P. aeruginosa undergoes alteration from the classical nonmucoid form to an atypical mucoid form, which correlates with the production of the exopolysaccharide alginic acid (6). In the context of chronic lung infection, alginate is an important virulence determinant. The appearance of alginate-producing P. aeruginosa strains is usually associated with a poor patient prognosis (10). Alginate has been implicated in adherence to tracheal epithelium (21, 28) and has been associated with antiphagocytic function (29). While the relationship between mucoid P. aeruginosa and CF is well documented, the environmental factors of the CF lung responsible for conversion of nonmucoid P. aeruginosa to the alginate-producing mucoid form have yet to be defined. A chemostat model for the study of this organism under chronic diseaselike growth conditions (phosphate and nitrogen limitation) which would reflect the likely physiological status of organisms in the lungs of CF patients has recently been reported (18). In this study, phosphate-limited cultures of mucoid P. aeruginosa were capable of utilizing phosphorylcholine (PC), a product of phospholipase C activity on phosphatidylcholine (a major component of lung surfactant), as a sole source of phosphate for growth (18). In an effort to understand the environmental factors responsible for the conversion of nonmucoid P. aeruginosa to the mucoid phenotype during chronic lung infection in CF patients, studies have been extended to investigation of the growth of nonmucoid P. aeruginosa in a chemostat system. In this report, prolonged continuous culture of nonmucoid P. aeruginosa PAO1 under several environmental conditions *
MATERIALS AND METHODS
Maintenance of bacteria. P. aeruginosa PAO1 was obtained from B. Iglewski, Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, N.Y. All stocks were maintained at -80°C in 10% skim milk and subcultured on mucoid maintenance agar (MMA) plates (MacConkey agar base modified by the addition of 50 g of glycerol per liter) (7). Culture media. A modification of the chemically defined alginate-promoting (AP) medium of Mian et al. (25), which has been described previously (17, 18), was the primary growth medium used for the chemostat studies. The medium contained 100 mM monosodium glutamate, 100 mM sodium gluconate, 7.5 mM NaH2PO4, 16.3 mM K2HPO4, and 10 mM MgSO4 7H20. The pH was adjusted to 7.0 with 2.5 N NaOH. For growth under various environmental conditions, further modifications of this medium were necessary. These modifications are summarized in Table 1. Continuous-culture conditions. Continuous culture was performed in a New Brunswick Bioflo bench-top chemostat (model C30; New Brunswick Scientific Co., Inc., Edison,
Corresponding author. 471
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TABLE 1. Growth media used for continuous culture of PAO1 Medium components AP' plus osmotic agent (0.3 M NaCl, 0.4 M sucrose, or 5 or 10% glycerol) High osmolarity ....................... AP plus monosodium glutamate reduced to 1 mM or replaced by 1 mM Nitrogen limitation (N medium) ....................... NaNO3 Carbon limitation (C medium) ....................... 2 mM K2HPO4, 18 mM NH4Cl, 10 mM MOPS,b 1 mM MgSO4 7H20, 2 mM carbon source, pH adjusted to 7.0 AP with NaH2PO4 and K2HPO4 replaced by 0.05 mM K2HPO4, 10 mM Phosphate limitation (P medium) ....................... MOPS added as a buffer, pH adjusted to 7.0 PC P medium with phosphate replaced by 10 mM PC chloride Phosphate source ....................... 2 mM K2HP04, 10 mM MgSO4 7H20, 100 mM sodium gluconate, 10 mM Nitrogen source ....................... MOPS, 1 mM PC chloride, pH adjusted to 7.8 Carbon source ....................... C medium with 2 mM PC chloride as sole carbon source Sole source of C, N, and P04 ..................... 10 mM PC chloride, 10 mM MgSO4 7H20, 10 mM MOPS, pH 7.0
Culture conditions
aAP, Alginate-promoting medium. MOPS, 3-[N-Morpholinolpropanesulfonic acid.
b
N.J.) with either a 350-ml or a 1.2-liter working volume. Culture conditions were as described previously (18) with the following modifications. When anaerobic conditions were required, nitrogen gas was supplied to the cultures at a rate of 0.5 liter min-', and sodium nitrate was added to the medium at a final concentration of 2% to serve as an alternative electron acceptor (31, 34). For continuous-culture studies which examined growth under conditions of high medium osmolarity, the addition of medium from the reservoir was initiated when the batch cultures reached the mid-exponential phase. All cultures were maintained at a specified dilution rate for several days. Growth and colony morphology. Samples were obtained from the culture vessel at various time points during growth of strain PA01. Appropriate dilutions were made in AP medium, and 0.1-ml aliquots of these samples were plated onto MMA plates for determination of growth based upon colony-forming units (CFU) per milliliter. The phenotypic expression of the culture was determined at the same time by noting the number of each colony type on MMA plates. Production of alginate by representative mucoid colonies was verified by isolation and purification of alginate by previously described methods (18), and the stability of mucoid colonies was determined by a minimum of three passages on MMA. Bacteriophage determinations. Plaque assays were performed with filtrates made from samples taken directly from chemostat cultures. Filtrates were diluted in phage suspension medium (SM) containing 100 mM NaCl, 10 mM MgSO4 .7H20, 0.1% gelatin, and 50 mM Tris, pH 7.5 (20). Aliquots (0.1 ml) were spread over 0.5% soft agar lawns of PA01. Following overnight incubation at 37°C, plaques were counted and phage activity was determined in plaque-forming units (PFU) per milliliter. Samples which contained phage activity were pooled and stored at 4°C for further use in phage lysate preparations. Phage stocks were made from plate lysates prepared from plaque-purified phage samples. Stocks were stored frozen in SM with 15% glycerol at -80'C or in SM at 4°C until used for phage sensitivity and adsorption assays. Phage sensitivity and adsorption assays. Nonmucoid colonies from plated samples of chemostat cultures taken at different times during the course of these cultures were screened for phage sensitivity. These colonies were subcultured first onto MMA plates. Only those colonies which did not spontaneously lyse after 48 h of incubation at 370C were tested for phage sensitivity. Colonies were resuspended in
AP medium and grown overnight in 50-ml cultures at 37°C with constant shaking. A 0.1-ml volume of each culture was spread onto MMA plates, which were allowed to dry for 60 min. Aliquots (50 ,u) of phage preparations were spotted onto these plates. After overnight incubation at 37°C, plates were checked for zones of clearing where phage had been spotted. Mucoid PAO1 variants isolated from cultures were treated in the same manner except that lawns were made in 0.5% soft agar in order to reduce the amount of exopolysaccharide production, which often obscured signs of phage activity. Plates from the original PA01 stock were also treated with phage preparations as a positive control. Mucoid variants were also tested for sensitivity by infection of soft-agar lawns with diluted phage samples and comparison with the number of plaques formed in nonmucoid PAO1 lawns infected with identical phage preparations. Mucoid variants were also compared with nonmpcoid PA01 in adsorption assays for their ability to adsorb phage. Triplicate samples of 108 to 109 cells from overnight cultures were mixed with an equal volume of SM containing 104 to 105 PFU of phage in microfuge tubes. After a 40-min incubation at 37°C, the mixtures were centrifuged at 1,100 x g for 15 min to get rid of cells, and supernatant fluids were subjected to sterile filtration. Samples were thep diluted in SM, and 0.1-ml volumes of each were spread onto soft-agar lawns of PAO1. Plates were incubated overnight, and plaques were counted. Phage adsorption was calculated from the reduction in the phage titer of samples compared with the titers of identical phage preparations which had not been exposed to cells. Statistically significant differences were assessed by a two-tailed Student's t test for independent means (P - 0.05 was considered not significant). RESULTS Osmotic stress. Growth of P. aeruginosa PAQ1 under conditions of high medium osmolarity was accomplished by continuous culture of organisms in AP medium with either 0.3 M NaCl or 0.4 M sucrose as osmotically active agents. Additionally, growth in AP medium with either 5 or 10% (vol/vol) glycerol, an osmotically neutral agent, was investigated. The results of these experiments were compared with those in continuous culture in AP medium alone (Table 2). Cultures were maintained for several days; however, it was difficult to achieve a steady state of growth as defined by constant CFUJ per milliliter on MMA plates for more than 48 to 60 h. Under all growth conditions, within the first 30 h of
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TABLE 2. Effect of increased osmolarity on mucoid conversion of P. aeruginosa PAO1 Osmotic agent
Concn
NaCI Sucrose Glycerol
0.3 M 0.4 M 5%
10% Nonec
D
% Mucoid
(h-1)
coloniesa
0.094 0.104 0.097
2.9
0.132 0.139
O.Ob
TABLE 3. Mucoid conversion of strain PAO1 during nitrogen or carbon limitation Growth conditiona
Nitrogen limitation Glutamate
3.5
5.6
Nitrate
O.Ob
a Percentage of colonies which expressed the mucoid phenotype at the end of the growth experiment. b No mucoid colonies detected during the growth experiment. I AP medium alone.
culture, there was a significant decrease in culture viability coincident with the appearance of two new and unusual colony morphotypes on MMA plates. One colony was of normal size but appeared to be undergoing lysis and completely disintegrated over time. The second colony type was small and slow growing and began to lyse within 48 h. Resuspension of these colonies in AP medium and inoculation of filtered supernatant fluids onto soft-agar lawns of PAO1 yielded bacteriophage plaques after overnight incubation at 37°C. Thus, it appeared that during the course of continuous culture of P. aeruginosa PAO1, a bacteriophage was produced. Following the appearance of the lysing colonies and evident bacteriophage activity, mucoid colonies appeared on MMA plates inoculated from cultures of PAO1 grown in the presence of 0.3 M NaCl, 5% glycerol, or 10% glycerol (Table 2). The percentage of the viable culture population which expressed the mucoid phenotype at the end of each growth experiment was 2.9, 3.5, and 5.6% for growth in 0.3 M NaCl, 5% glycerol, and 10% glycerol, respectively. No mucoid variants were detected when 0.4 M sucrose was used as the osmotic agent or when strain PAO1 was grown in AP medium alone (Table 2). Nitrogen limitation. Glutamate, a rapidly assimilated nitrogen source, and nitrate, a poorer source of nitrogen, were chosen for the investigation of the effect of nitrogen limitation on mucoid conversion of P. aeruginosa PAO1. Growth in N medium with 1 mM glutamate as the nitrogen source yielded no mucoid organisms, even at very slow growth rates. Conversely, at slower growth rates in cultures supplied with nitrate, a subpopulation of mucoid variants was detected (Table 3), with similar cultures yielding from 0.1 to 0.4% of viable organisms expressing the mucoid phenotype on MMA plates. High bacteriophage titers were observed in all cultures, but only cultures grown with nitrate as the sole nitrogen source produced mucoid organisms (Fig. 1). Carbon limitation. Four carbon sources, acetate, glycerol, succinate, and gluconate, were chosen for carbon limitation experiments. Cultures were maintained at low dilution rates (D), which ranged from 0.038 to 0.058 h-'. Again, high titers of bacteriophage were detected during each experiment. Gluconate-limited growth failed to yield mucoid PAO1 variants. In contrast, when the growth-limiting carbon sources were composed of fewer carbons, mucoid organisms appeared consistently. For acetate, glycerol, and succinate, the level of mucoid organisms was 2.8, 1.3, and 4.4%, respectively (Table 3). Figure 2 illustrates a comparison between growth in gluconate- and growth in succinatesupplemented C medium (Fig. 2A and B, respectively). Interestingly, 3% of the succinate-grown organisms were
473
Carbon limitation Acetate Glycerol Succinate Gluconate
D
% Mucoid
(h-')
coloniesb
0.032 0.353 0.046 0.045
0.OC 0.Oc
0.058 0.046 0.046 0.038
2.8 1.3 4.4
0.4 0.1
0.0O
a For nitrogen limitation, 1 mM nitrogen source; for carbon limitation, 2 mM carbon source. b Percentage of colonies which were mucoid at the end of the growth
experiment. ' No mucoid colonies detected during culture period.
mucoid almost 24 h before any phage activity was detected (Fig. 2B). Effect of growth rate on phenotypic expression in phosphate-limited cultures. The effect of decreased growth rates on nonmucoid organisms, as might be found during prolonged chronic P. aeruginosa lung infection, was examined in phosphate-limited cultures of strain PAO1. Dilution rates (D) ranged from 0.035 to 0.347 h-1. Mucoid variants were detected only when D was below 0.347 h-1. The percentage of mucoid organisms in cultures ranged from 0.8% at a D of 0.173 h-1 to 40% at a D of 0.044 h-1 (Table 4). High titers of bacteriophage were detected under all conditions. However, these results were independent of bacteriophage activity, as cultures grown at a relatively high D of 0.347 h-1 (Table 4) or grown anaerobically yielded no mucoid organisms (data not shown). Phenotypic expression in cultures supplied with PC as the limited nutrient source. Continuous culture of strain PAO1 with a growth-limiting amount of substrate likely to be found in the lungs was investigated. PC, used previously in phosphate limitation studies with mucoid organisms (18), was used as the growth-limiting phosphate, nitrogen, or carbon source. Also, this compound was evaluated as the sole source of carbon, nitrogen, and phosphate. Growth of phosphate-limited organisms supplied with 10 mM PC as the phosphate source resulted in expression of the mucoid phenotype by 0.35% of the viable organisms by the end of the culture period (Table 5). Mucoid colonies were evident by day 2 of culture, following the appearance of bacteriophage activity. When PC was supplied as the limiting nitrogen source, 0.49% of the population was mucoid by the end of the culture period (Table 5). Mucoid colonies were detected almost 24 h before phage activity. Cultures of strain PAO1 supplied with PC as the growth-limiting carbon source or as the sole source of carbon, nitrogen, and phosphate yielded 0.1 and 0.88% mucoid organisms (Table 5). Mucoid colonies were observed more than 48 h prior to detectable phage activity in the latter culture. Significance of bacteriophage in PAO1 continuous cultures. Although high levels of bacteriophage were produced under all growth conditions in continuous cultures of nonmucoid PAO1, mucoid variants were observed only when cultures were grown at a relatively low D and only in the presence of high salt or high glycerol (0.3 M NaCl or 5 or 10% glycerol, Table 2) or under limitation for specific nutrients (Tables 3 to
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FIG. 1. Growth and phenotypic expression of P. aeruginosa PA01 during nitrogen-limited growth in N medium with (A) glutamate and (B) nitrate as the nitrogen source. Symbols: 0, viable organisms; 0, phage activity. The percentage of the culture population which expressed the mucoid phenotype is shown by solid bars in the graph below each growth curve.
5). Additionally, screening of colonies isolated from chemostat cultures revealed that the culture population, both mucoid variants and nonmucoid PAO1, remained sensitive to this phage. Isolation of phage-resistant variants of PAO1 was successful only early in cultures, during the initial burst of phage activity when the cultures went into decline. Thereafter, this subpopulation of the cultures became undetectable as the cultures recovered toward more stable growth. Phage-resistant variants grown under phosphatelimited conditions which yielded 40% mucoid organisms from the original PAO1 culture yielded much lower levels of mucoid organisms. However, these variants have not been characterized further with respect to differences from PAO1 other than phage resistance. Mucoid PAO1 variants were tested for phage sensitivity, and all proved to be sensitive even though phage adsorption assays showed significantly lower adsorption of this phage to mucoid variants than to nonmucoid PAO1 (data not shown). Finally, phage production in batch cultures was investigated. Phage could only be detected in cultures inoculated from MMA plate cultures which were 1 week or more in age. These cultures did not yield mucoid variants. DISCUSSION
Mucoid variants of P. aeruginosa have been recovered from chronic lung infections associated with CF and chronic obstructive lung disease as well as from chronic urinary tract infections (13). More recently, mucoid variants of P. aeruginosa PAO were isolated from the lungs of chronically infected rats (32). Speert et al. (30) have reported isolation of mucoid organisms from prolonged static cultures in acetamide broth. Therefore, conversion to mucoidy is not unique to the CF lung but may take place in response to common
environmental factors encountered during chronic infection or in vitro under adverse growth conditions. The environmental triggers for mucoid conversion are not yet understood. The purpose of this investigation was to determine the environmental conditions conducive to mucoid conversion during continuous culture of a nonmucoid strain. Experiments were designed to reflect conditions which might be present during chronic infection of the CF respiratory tract. One environmental factor relevant to CF is osmotic stress. Exocrine secretions in CF contain elevated sodium, chloride, and calcium ion levels (16), which would result in increased osmolarity of bronchial secretions. Growth of P. aeruginosa PAO1 in 0.3 M NaCl resulted in conversion of 2.9% of the organisms to mucoidy. Enhanced transcription of the biosynthetic algD gene has been reported under similar conditions (2). Ionic strength may be more important than the osmotic component, however, since no mucoid variants were detected during growth in 0.4 M sucrose. Similar differential effects have been noted for amino acid uptake in osmotically stressed Escherichia coli (9). Growth in 5 or 10% glycerol, an osmotically neutral agent, also resulted in isolation of mucoid variants. Excess glycerol can cause formation of toxic metabolites (8, 19). Perhaps these mucoid variants were derived from a subpopulation of organisms more acutely sensitive to this metabolic stress. A second environmental factor would be nutritional limitation. Chronic persistence of P. aeruginosa on mucosal surfaces in the lungs without extensive tissue invasion would require prolonged survival in an environment limited for nutrients. Limitation of nutrients would greatly affect the physiological status of P. aeruginosa, particularly intracellular energy metabolism. Growth rates would have to decrease and organisms would need to become more efficient at utilizing limited nutrients. The results of the phosphate
MUCOID CONVERSION OF P. AERUGINOSA PA01
VOL. 59, 1991
A
475
B
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FIG. 2. Phenotypic expression of P. aeruginosa PAO1 during carbon-limited growth in C medium with (A) gluconate and (B) succinate the carbon source. Symbols are the same as in Fig. 1.
limitation experiments are consistent with this idea. At the highest dilution rate (0.347 h-1), mucoid variants were not seen. At very low dilution rates (0.035 to 0.044 h-1), cultures had access to phosphate concentrations well below the 1 mM level known to induce genes responsive to phosphate deprivation in E. coli (23). Phosphate limitation has a direct impact on intracellular energy pool levels, which is probably the reason for the relationship observed between growth rate and the percentage of mucoid organisms in these cultures. The results of the carbon and nitrogen limitation experiments also indicate a relationship between energy metabolism and mucoid conversion. Carbon-limited organisms grown on substrates other than gluconate would experience a greater energy drain due to the increased demand for more tricarboxylic acid cycle intermediates for biosynthetic reactions. No mucoid organisms were isolated when gluconate was used as the carbon TABLE 4. Effect of growth rate on mucoidy in phosphatelimiteda cultures of PAO1 D (h-1)
% Mucoid coloniesb 38.6 0.035 ..................................... 40.0 0.044 ..................................... 2.0 0.069 ..................................... 1.0 0.116 ..................................... 0.8 0.173 ..................................... 0.0 0.347 ..................................... a 0.05 mM K2HPO4. b Percentage of colonies which were mucoid at the end of the growth experiment. c No mucoid colonies detected during the culture period.
source. Likewise, nitrate assimilation requires a larger expenditure of potential energy than glutamate assimilation, in the form of reducing equivalents (27). Mucoid organisms were only detected when the limited nitrogen source was nitrate. Changes which occur during nutritional limitation or starvation have been studied in various organisms (14, 23). Exopolysaccharide synthesis has also been demonstrated during starvation stress (33). The results from our studies with P. aeruginosa PAO1 suggest that mucoid variants may first arise during chronic infection in response to nutrient limitation. Once these variants appear, alginate would afford them protection from host defense mechanisms such as phagocytosis (29), allowing them to persist. The lung may offer a variety of alternative nutrient sources which are liberated by the action of P. aeruginosa exoenzymes. PC is liberated from the major lung surfactant phospholipid phosphatidylcholine by the action of P. aerug-
TABLE 5. Mucoid conversion in PAO1 cultures supplied with PC as an alternative nutrient source Limited nutrienta
Carbon
Nitrogen Phosphate C, N, and P04
D
% Mucoid
(h-1)
coloniesb
0.046 0.044 0.111 0.059
0.10 0.49 0.35 0.88
a For carbon, nitrogen, phosphate, and combined (C, N, and P04) limitation, PC was used at 2, 1, 10, and 10 mM, respectively. b Percentage of colonies which expressed the mucoid phenotype at the end of the culture period.
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inosa phospholipase C (1). Phosphate-limited cultures of mucoid organisms have been shown to utilize this substance for alginate production and as a source of phosphate for growth (18). PC offers P. aeruginosa the phosphate, carbon, and nitrogen needed for growth. Mucoid variants of PAO1 were isolated from carbon-, nitrogen-, and phosphate-limited cultures supplied with PC as the sole source of these nutrients. Thus, the environment of the lung offers a potential growth substrate for P. aeruginosa, and utilization of this substrate for growth can yield mucoid variants. While nutrient limitation experiments resulted in isolation of mucoid variants of PAO1, all growth conditions also resulted in production of a bacteriophage associated with this organism. Whether the bacteriophage which was detected was a clear-plaque mutant of a resident lysogenic phage or was a phage existing in a pseudolysogenic state is not yet known. However, the phage was able to infect and lyse strain PAO1. Although phage-mediated mucoid conversion has been reported (22, 26), our results suggest that environmental conditions regulated the appearance of mucoid variants. All mucoid variants tested proved to be sensitive to this phage. Nonmucoid phage-resistant variants of PAO1 were only detected in chemostat cultures during the period of the initial burst of phage activity, when these cultures went into decline. During recovery of these cultures and the return to more stable growth, this resistant subpopulation became undetectable. If the phage was a significant selective agent, selection of resistant organisms, which would predominate over sensitive subpopulations, would be expected. At no time was this the case. Clearly, the results of these studies with P. aeruginosa PAO1 indicate that many environmental factors can play a role in mucoid conversion. The conversion process is not an all-or-nothing response by organisms to environmental stresses but seems to be regulated by the degree to which subpopulations of cells are sensitive to those stresses. There are several widely dispersed genes involved in regulation and synthesis of alginate (3, 5, 11, 12). In nature or in chronic infection, P. aeruginosa must adapt to several environmental stresses. The regulatory algR gene may be part of a sensory transduction circuit responsive to one or more environmental signals which trigger activation of algD and other alg biosynthetic genes (4). The roles of other regulatory genes are not yet clear. Isolation of mucoid variants by continuous-culture methods which allow control of environmental conditions without the use of mutagenic agents is a strategy which might be useful in this pursuit. Analysis of mucoid variants obtained in this way will help in the determination of the genes involved in the complex regulation of alginate synthesis. ACKNOWLEDGMENTS We thank John J. Maurer for his valuable technical assistance and John Govan and Vojo Deretic for their many helpful discussions. This research was supported by grant Z070 from the Cystic Fibrosis Foundation. J.M.T. was supported, in part, by Public Health Service training grant T32 AI-07271 from the National
Institutes of Health.
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