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Arginine deiminase system and bacterial adaptation to acid environments. R E Marquis, G R Bender, D R Murray and A Wong Appl. Environ. Microbiol. 1987, 53(1):198. Updated information and services can be found at: Downloaded from on February 24, 2012 by guest These include: CONTENT ALERTS Receive: RSS Feeds, eTOCs, free email alerts (when new articles cite this article), more» Information about commercial reprint orders: http://aem.asm.or
   1987, 53(1):198. Appl. Environ. Microbiol. R E Marquis, G R Bender, D R Murray and A Wong adaptation to acid environments.Arginine deiminase system and bacterial information and services can be found at: These include:  CONTENT ALERTS  more»cite this article),Receive: RSS Feeds, eTOCs, free email alerts (when new articles Information about commercial reprint orders: To subscribe to to another ASM Journal go to:  onF  e b r  u ar  y 2 4  ,2  0 1 2  b  y  g u e s  t  h  t   t   p:  /   /   a em. a s m. or  g /  D  ownl   o a d  e d f  r  om   Vol. 53, No. 1 PPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1987, p. 198-200 0099-2240/87/010198-03$02.00/0 Copyright ©) 1987, American Society for Microbiology Arginine Deiminase System and Bacterial Adaptation to Acid Environments ROBERT E. MARQUIS,* GARY R. BENDER,DANIEL R. MURRAY, AND ALAN WONG Department of Microbiology, University of RochesterSchool of Medicine and Dentistry, Rochester, New York 14642 Received 14 August 1986/Accepted29 September 1986 The arginine deiminase system in a variety of streptococci and in Pseudomonas aeruginosa was found to be unusually acid tolerant in that arginolysis occurred at pH values well below the minima for growth and glycolysis. The acidtoleranceofthe system allowed bacteria to survive potentially lethal acidification through production of ammonia to raise the environmental pH value. The arginine deiminasesystem provides a source of ATP derived from catabolism of arginine to ornithine, C02, and NH3 in a variety of organisms, including many streptococci and members of the genus Pseudomonas (1). The system is generally inducible andunder the control of catabolite repression. It appears to play a role in allowing the predom- inantly aerobic Pseudomonas organisms to grow anaerobi- cally without respiration (12). We have recently isolated strains of Streptococcus faecium and S. sanguis that have defective cataboliterepres- sion (3, 5) and cansimultaneouslydegrade arginine and glucose. Initial assessments of the acid sensitivity of arginine catabolism by these organisms revealedan unexpected de- gree of acidtolerance, which is also shown by the parent strains. In effect, it appears that arginine can be catabolized at pH values well below the minima for growth or glycolysis. Organisms chosen for further study include S. faecium ATCC 9790, S.lactis ATCC 19435, S. rattus BHT and ATCC 19645, S.milleri ATCC 9895, S. sanguis ATCC 10556, Challis, and NCTC 10904, and Pseudomonas aerugi- nosa (ATCC 15442). They weremaintained on tryptone- glucose-Marmite (Marmite Ltd., Montreal, Quebec,Can- ada) agar with weekly transfers and also as lyophilizedpreparations. Cultures weregrown statically at 37°C in tryptone-Marmite medium (9) with added glucoseor argi- nine. Ammonia producedby thebacteria was assayed first by the Conway microdiffusion method (4) and subsequentlywith an ammonia electrode (OrionResearch, Inc., Cam- bridge, Mass.)connected to an Orion lonanalyzer (model 407A) by the procedures recommended by the manufacturer. ApH meter (model 45; Beckman Instruments, Inc., Fullerton, Calif.) connected to a glass electrode was used to determine pH values. Cell dryweights were determined bywashing (oncewithdeionized water) cells centrifuged fromsuspensions or cultures. The washed cells weresuspended in deionized water, and aportionof the suspension was placed in atared aluminum weighing panand dried to a constant weight at 95°C. The datapresented in Fig. 1 for S. faecium ATCC 9790 indicate that the bacteriumcandegrade arginine to produce ammonia at nearly constant pH values as low as 3.5. In these experiments, the pH value was heldnearlyconstant by intermittent addition of acid. This minimum pH value for * Corresponding author. arginolysis is nearly 1.5 pH units below the minimum for growth of the organism in complex medium and nearly 1 unit below the minimum for glycolysis (10). The rate of arginolysis at the low pH valueof 3.5 was less than that at pH valuesof 4.0 or 6.0, butthe bacterium could still degrade arginine in acid environments,whichhavebeen found to be damaging to the cell membrane, as reflected inloss of magnesium and metabolite pools (9). 15 pH 0 6.0 0 4.0 10 5 3.5 3.0 0 2040 60 MINUTES FIG. 1. Acid sensitivity of the arginine deiminasesystem of S. faecium ATCC 9790. Cultures weregrown to stationary phase in tryptone-Marmite broth containing 13.9 mM glucose plus 47.5 mM arginine.Cells were harvested by centrifugation, washed with water, andsuspended in 20 mM potassiumphosphate buffer with 1.0 mM MgCl2 at the indicated pH values to yield suspensions with 5 mg of cell dry weight per ml. Argininewasadded to yield a final concentration of 47.5 mM, and ammonia was assayed by means of an ammonia electrode. HCI solution was added to maintain nearly constant pH. Incubation was at 37°C. 198   onF  e b r  u ar  y 2 4  ,2  0 1 2  b  y  g u e s  t  h  t   t   p:  /   /   a em. a s m. or  g /  D  ownl   o a d  e d f  r  om   TABLE 1. Rise in pH associatedwith arginolysisa S. rattus S. rattus S. sanguis S. sanguis S. sanguis S. faecium S. lactis S. milleri P. aeruginosa Time BHT FA-1 NCTC 10904 ATCC 10556 Challis ATCC 9790 ATCC 19435 ATCC 9895 ATCC 15442 (h) NH3 pH NH3pHNH3pH NH3 NH3 pH NH3 pH NH3 pH NH3 H NH3 pH ~Lmol/ ) pH ( l/l)mol/ml) 4LMOI) (ILLmol/mpH N(,mol/ml) 4Lmol/ml) (,umol/ml) (sLmol/ml) 4Lm ) 00.104.00 0.10 0.704. 4.00 1 0.24 4.15 1.244.50 0.714.40 1.00 4.70 5.005.70 20.006.70 4.0030.76 7.60 2 1.104.70 3.205.35 35.00 7.20 43.007.50 1.584.25 0.094.0036.607.90 3 1.405.0033.00 6.852.285.20 5.10 5.55 44.00 7.45 64.007.80 2.934.75 0.09 4.0041.72 8.15 4 2.105.3043.007.30 3.705.25 8.305.7055.00 7.75 64.00 7.853.635.421.98 4.2044.20 8.25 5 6.405.6061.00 7.60 6.405.6015.506.1569.00 7.8564.00 7.95 5.50 5.95 2.394.6053.32 8.25 6 7.205.7080.007.90 10.40 5.95 29.00 6.6080.00 8.00 66.00 8.0014.676.552.64 4.9063.76 8.20 24 27.507.4080.00 7.90 46.00 7.40 46.007.6080.00 8.20 82.00 8.00 55.65 7.95 23.76 6.75 66.32 8.10 a Cultures were grcwn in tryptone-Marmite broth plus 5.6 mM glucose and harvested at the mid-exponential phase.After being washed, the cells were suspended at a density of 5 mg (dry weight)per ml in 20 mM potassiumphosphate buffer with 1.0 mM MgCI2 at a pH of 4.0. Arginine was added to47.5 mM final concentration, and the suspensions were incubated at 37°C. Arginolysis by the other bacteria we tested wassomewhat less acid tolerant than that by S. faecium ATCC 9790. However, each of the organisms was able to degrade argi- nine at an initial pH of 4.0 (Table 1), or approximately the minimum pH of dental plaque (11). In these experiments, the pH was notheld constantbut rose to values above 7asthe bacteria produced ammonia. There was not a close corre- spondencebetween total ammonia producedand final pH, partly because of differences in buffering by the different types of cells, but mainlybecause some of the bacteria had storage polymers which can bedegraded to produce acid end products. However, in all cases,the capacity to produce ammonia from arginine was greater than the capacity to produce acid from endogenous reserves. Inaddition, when thebacteria were in growth media, they could start to grow after the pH value had risen to the minimum for growth. In this sort of experiment with rising pH values, S. faecium ATCC 9790could degrade arginine at anextremely low initial pH of 2.5(Fig. 2) (however, the pH was only this low initially). At 24 h, the pH had risen to nearly 8.0 and the bacteria had increased the NH3 level to nearly 80 mM. In similar experiments, P. aeruginosacould produce ammonia atinitial pH values as low as 3.0. The other bacteria tested were less acid tolerant in regard to argininecatabolism, but could produce ammonia at initial pH valuesof 4.0 or somewhat lower. For the experiments described in Table 1, cells were harvested from cultures grown in medium with a high level of glucose (55.6 mM) and no added arginine. These cells were repressed for the arginine deiminase system, although with this system repression is not complete (5). The re- sponses of the cells thus included not only initial arginine degradation but also subsequent derepression. Although extremely acidic environments can be lethal, it appeared that catabolism of arginine with a resultant risein pH could spare the test organisms. For example, when cells of S. faecium ATCC 9790 were suspended in 20 mM potas- sium phosphate buffer with 1 mM MgCl2 to give a suspension of 1.9 x 109 cells per ml at a pH value of 2.5, the cells remained viable for 1 h at room temperature (as indicated by counts of spread plates prepared with Trypticase soy agar [BBL MicrobiologySystems, Cockeysville, Md.] at pH 7.0 and incubated at 37°C for 48hbefore the colonies were counted). However, after 1 h, there was approximately a 10-fold drop in viable count perh until 6 h, when the count had declined to 2.0 x 105 cells per ml; the pH remained at 2.5. In contrast, when 47.5 mM arginine was added to a companion suspension, there was essentially no decrease in viability over the6-h period, and the pH rose to 7.9. At an initial pH value of 2.0, the population was completely eliminated after 1 hof incubationwithout arginine. Although addition of arginine slowed the killing (at 5 h some 1.04 cells per mlremained alive), all of the cells still died. At pH valuesof 3.0 or 4.0, death occurred more slowly in populations without arginine, with only a 10-fold reduction in numbers after 6 h. Comparable suspensions to which arginine was 80.0 60.0 --j E 0 E :1. 1- Cl) zz 40.0 20.0 0 1 2 3 4 5 6 24 HOURS FIG. 2. Ammonia production at low pH values by S. faecium ATCC 9790. Cultures were grown in tryptone-Marmite broth plus 55.6 mM glucoseandharvested at mid-exponential phase. Other experimental details are described in the legend to Fig. 1, except that HCI solution was not added to maintain a nearly constant pH. VOL. 53, 1987 NOTES 199   onF  e b r  u ar  y 2 4  ,2  0 1 2  b  y  g u e s  t  h  t   t   p:  /   /   a em. a s m. or  g /  D  ownl   o a d  e d f  r  om   APPL. ENVIRON.MICROBIOL. added showed no reduction in cell numbers and an increase in pH to 7.9 at 6 h. In conclusion, we found that the arginine deiminase sys- tem can playan important role in the acid-base physiologyof each of thebacteriastudied in that it could allow for recovery from acid stressessufficiently severe to stop growthand glycolysis. For example, our past work (2, 8) indicated that the minimum pH values forglycolysis by S. faecium ATCC 9790 and S. rattus FA-1 in cultures are 4.4 and 4.8, respectively, while minimum pH values for growth are 4.8 and 5.4. However, S. sanguis NCTC 10904 proved to be less acid tolerant, with minimum pH values ofabout 5.2 for growth and glycolysis; yet all of these bacteria, and the others tested, were able to carryout arginolysis at a pH of 4.0 and to raise the pH value above neutrality. Kanapka and Kleinberg (7) have found that organisms in salivary sediment degrade arginine optimally at pH values between 7.0 and 8.0 and that there is low-level activity at a pH of 4.0. The mixtures they used contained 2.8 mM glucose, and the sugar may have suppressed arginolysis. Overall, it seems that streptococci having the arginine deiminase system are adapted to reverse the adverse effects of acid environments. Even when streptococci such as S. sanguis and S. faecium are grown in media with repressive levels of glucose, there still is production of ammonia anddegradation of arginine during thestationary phase when the pH value is low (3). Thistypeofderepression is probably best interpreted in termsof reduction in the level of the glucose-specific phosphotransferasesystem(PTS) at low pH values as shown, for example, by Hamilton and St. Martin (6). Enzyme II of the glucose-PTS appears to be the major effector of cataboliterepression, atleastin oral streptococci (G. R. Bender and R.E. Marquis, J. Dent. Res. 65:242, abstr. no. 653,1986). At low pH values,the glucose-PTS is repressed; hence, catabolite repression is reducedand am- monia is producedfrom arginine. If there is sufficient argi- nine available, the pH value willrise (Table 1), and glycolysis andgrowth canbe reinstated. Of course, once the glucose-PTS hasagain started to function, if there is avail- able glucose, ammonia production will be repressed, and arginine can be spared for possible useduringanother acid stress. This work was supported by Public Health Service grant RO- DE06127from the National Institute of Dental Research and grant PO1-DE07003 from the Rochester Cariology Center. LITERATURE CITED 1. Abdelal, A. T. 1979. Argininecatabolism in microorganisms. Annu. Rev. Microbiol.33:138-168. 2. Bender,G. R., E. A. Thibodeau, and R.E. Marquis. 1985. Reduction ofaciduranceof streptococcal growthand glycolysis by fluoride and gramicidin. J. Dent.Res. 64:90-95. 3. Campbell, J., III, G. R.Bender, and R. E. Marquis. 1985. Barotolerant variant ofStreptococcus faecalis ATCC 9790with reduced sensitivity to glucose cataboliterepression. Can. J. Microbiol. 31:644-650. 4. Conway, E. J. 1962. Microdiffusion analysis and volumetric error. Crosby Lockwood and Son, London. 5. Ferro, K. J., G. R. Bender, and R. E. Marquis. 1983. Coordi- nately repressible arginine deiminase system in Streptococcus sanguis.Curr.Microbiol.9:145-149. 6. Hamilton, I. R., and E. J. St. Martin. 1982. Evidence for the involvement of proton motive force in thetransport ofglucose by a mutant of Streptococcus mutans strain DR0001 defective in glucose-phosphoenolpyruvate phosphotransferase activity. In-fect. Immun. 36:567-575. 7. Kanapka, J. A., and I. Kleinberg. 1983. Catabolism of arginine by the mixed bacteria in human salivary sedimentunder condi- tions of lowand high glucoseconcentration.Arch. Oral Biol. 28:1007-1015. 8. Marquis, R. E., and G. R. Bender. 1980.Isolation of a variant of Streptococcus faecalis with enhanced barotolerance. Can. J. Microbiol.26:371-376. 9. Marquis, R. E., N. Porterfield, and P. Matsumura. 1973. Acid- base titration of streptococci and the physical states of intracel-lular ions. J. Bacteriol. 114:491-498. 10. Matsumura, P., D. M. Keller, and R.E. Marquis. 1974. Re- stricted pH ranges andreduced yieldsforbacterial growth under pressure. Microb. Ecol. 1:176-189. 11. Schachtele, C. F., and M. E.Jensen. 1982. Comparison of methods for monitoring changes in the pH of human dental plaque. J. Dent. Res.61:1117-1125. 12. Vander Wauven, C., A. Pierard, M. Kley-Raymann,and D.Haas. 1984. Pseudomonas aeruginosamutants affected in anaerobic growth on arginine: evidence for a four-gene cluster encoding the arginine deiminasepathway. J. Bacteriol. 160: 928-934. 200 NOTES   onF  e b r  u ar  y 2 4  ,2  0 1 2  b  y  g u e s  t  h  t   t   p:  /   /   a em. a s m. or  g /  D  ownl   o a d  e d f  r  om 
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