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Extracellular product(s) of Staphylococcus aureus stimulate their own growth
Zentralblatt fUr
Zent.bl. Bakteriol. 289, 339-345 (1999) © Urban & Fischer Verlag http://www.urbanfischer.de/j ournals/ zblbakteriol
Extracellular Product(s) of Staphylococcus aureus Stimulate Their Own Growth Noriaki Sakata\ Yukio Usui h
,
and Tadahisa Kogure 1
Department of Legal Medicine and Department of Microbiology, St. Marianna University School of Medicine, Kawasaki 216-8511, Japan * Present address; Laboratory of Nutrition and Biochemistry, Aoba-Gakuen Junior College, Tokyo
1
2
Received October 5,1998 . Revision received February 10, 1999 . Accepted February 26, 1999
Summary The effect of extracellular products obtained from culture supernatant of Staphylococ cus aureus strain Cowan I on the bacterial growth was studied in a synthetic medium. Addition of the extracellular products to a fresh medium stimulated growth already after 2 h of incubation, with an approximately two-fold increase in cell density as com pared to an un supplemented medium, probably by promoting an initiation of growth accompanied by a reduction of the initial lag phase. The growth-stimulating effect was also monitored as an increase of adenosine triphosphate (ATP) in the bacterial culture during the different phases of growth.
Introduction Many kinds of cellular factors which regulate various cell functions and main tain cooperation between cells in various tissues are well designed in higher multicellular organisms. Even in prokaryotic organisms such as bacteria, intercellular signals seem to mediate cell-cell communication (10). For in stance, several intercellular signals are involved in cell differentiation and morphogenesis, such as fruiting body formation in Myxococcus, erection of aerial hyphae in Streptomyces and the sporulation process in Bacillus (10). Similar systems which are involved in growth have also been suggested to exist 0934-8840/99/289/3-339 $12.00/0
340
N. Sakata, Y. Usui, and T. Kogure
in Francisella (Pasteurella) tularensis, Micrococcus luteus and Xanthomonas maltophilia (4, 6, 7, 8, 11, 12). Considering these examples, such systems of growth regulation through intercellular signals seem to be universal also in the bacterial world. Staphylococcus aureus is a major human pathogen causing a variety of dis eases. This gram-positive coccus can rapidly develop resistance to a variety of antibiotics. Provided such a self-promoting system of cell growth functions also in this bacterial pathogen, new non-antibiotic strategies to prevent and break these infections may be developed. Therefore, we examined a possibil ity that S. aureus produced an autocrine-like factor which was involved in growth regulation.
Materials and Methods Media
Medium 199 (MI99) and RPMI Medium 1640 (RPMI 1640) were purchased from GIBCO BRL, Life Technologies, Inc. (Rockville MD, USA). Brain heart infusion agar (BHI agar) was purchased from Difco Laboratories (Detroit, MI, USA). Culture conditions and preparation of S. aureus extracellular products
One loopful (1 fll) of S. au reus strain Cowan I on BHI agar was inoculated into 10 ml of M199 medium and cultured for 3 h at 37°C. The culture was inoculated into 400 ml of the liquid M199 medium and continued to culture for an additional 18 hat 37°C. The supernatant was obtained by centrifugation (11 000 xg for 20 min at 4°C) and stored at -80°C after filtration by 0.22 flm membrane filter (Millipore corpora tion, Bedford, MA, USA). The stored supernatant was dialyzed against distilled water by using a membrane with 1 kDa of molecular weigth cutoff (Spectrum, Houston, TX, USA) to eliminate the components included in the medium, and then lyophilized. The residue was dissolved in phosphate-buffered saline (PBS) and stored at -20°C. Determination of bacterial cell numbers
Separate standard curves for liquid media were utilized as a reference to determine bac terial cell numbers; the absorbance at 420 nm of a diluted bacterial suspension in the M199 and RPMI 1640 media with 0.1 volume of 1M Tris-HCI, pH 7.5, was measured and duplicate aliquots were plated on BHI agar plates to count bacterial colonies after 24 h incubation at 37°C. Effect of the extracellular products on bacterial growth
One loopful (1 fll) of S. au reus strain Cowan I on BHI agar was grown in 20 ml of M199 for 16 h at 3rC and the cells were washed five times with 40 ml of PBS. The washed cells were cultured, with a density of 3 X 106 cells/ml, at 3rC in M199 sup plemented with 50 flg of protein, or unsupplemented with the extracellular products.
After the specified period of incubation, 1 ml aliquots of culture were mixed with 100 fll of 1M Tris-HCI, pH 7.5, and the absorbance at 420 nm was measured by using M199 with 0.1 volume of 1M Tris-HCl, pH 7.5, as a reference standard.
Growth enhancement of S. aureus
341
Measurement of bacterial ATP during growth A luciferin-Iuciferase assay to determine bacterial ATP was performed in a LUMI COUNTER 700 (MICROTEC NITI-ON, Funabashi, Chiba, Japan) using the follow ing reagents: ATP releasing reagent (TURNER DESIGNS, Mountainview, CA, USA), luciferin-Iuciferase solution (MICROTEC NITI-ON) and HEPES buffer (MICROTEC NITI-ON). Briefly, the bacterial culture was grown overnight in the M199 medium at 37°C and cells were washed five times with PBS. The cells (3 X 10 7 ) were cultured at 37°C in 1.5 ml of RPMI 1640 supplemented with 50 fl.g of protein per ml, or unsup plemented with the extracellular product. 100 fl.l HEPES buffer, was added to a plas tic tube (MICROTEC NITI-ON) including 100 fl.1 of a bacterial culture after different periods of incubation and the mixtures were further incubated with 100 fl.1 of the ATP releasing reagent for 10 seconds. After incubation, 100 fl.l of luciferin-Iuciferase solu tion was added to the mixture and luminescence was measured for 10 seconds. Protein assay A protein assay was performed with BCA protein assay reagents (PIERCE, Rockford, IL, USA), using BSA as a standard.
Results and Discussion In this experiment, an enriched complex culture medium such as BHI seemed to be unsuitable for isolating extracellular products of S. au reus and investi gating their effects because it might contain various nutritional factors which masked the effects of the bacterial products by promoting or repressing the bacterial growth. Therefore, a synthetic medium was used in this study. Espe cially, the M199 medium, originally used for culturing mammalian cells, was considered to be expedient because it contained several vitamin essential for staphylococci, e. g. folic acid and p-aminobenzoic acid, besides amino acids and glucose. S. aureus strain Cowan I grew satisfactorily in this medium (data not shown) and bacterial culture supernatants were obtained (see Materials and Methods). The protein concentration of the culture supernatant was about 30 flg per m!. The effect of the extracellular products on bacterial growth was investigated by culturing S. aureus strain Cowan I in this medium supplemented with the products (Fig. 1). Preparation of bacterial cells for this experiment was also performed by using the M199 medium to eliminate a contamination by exo geneous substances from the culture medium. When staphylococci were incu bated with the extracellular products (50 flg of protein per ml) the cell density was enhanced 2.S-fold after 2 h of incubation, as compared to an unsupple mented medium. The difference in cell density was still maintained after 4 h of incubation. A medium supplemented with bovine serum albumin (BSA) was used as a control to explore the possibility that the presence of proteins might nonspecific ally affect bacterial growth. Addition of BSA (50 flg/ml) seemed to slightly enhance the cell numbers up to 2 h of incubation. However, this effect
342
N. Sakata, Y. Usui, and T. Kogure
of BSA was faint and cell numbers of the 3 h culture were almost equal to those of unsupplemented M199. Living bacterial cells contained ATP levels which were in relation to the cell mass (15). A growth enhancement as described above was confirmed by meas uring total ATP in the culture during bacterial growth (TableI). RPMI 1640,
E ~
Q) ~
Qj
.0
E
10 7
::J
C
iii
'55
'0 III m
10 6
0
4
23 Incubation period (h)
Fig. 1. Growth of S. aureus strain Cowan I in medium supplemented or non supplemented with the extracellular products. The bacterial cells were inoculated into the M199 medium supplemented (closed circle) or non-supplemented (open circle) with the bacterial extracellular products (50 [.tg of proteins/ml), with a density of 3 X 106 cells/ml. The medium supplemented with BSA (50 [.tg/ml open triangle) was also used as a control. The bacterial numbers were determined as described in Materials and Methods. Values are means of duplicate determinations.
Table 1. Total amount of ATP in bacterial culture during growth Relative Light Units (RLU)
Medium Without Cells RPMI1640 0.018 RPMI1640+BSA 0.020 RPMI 1640 + Products 0.026
X
10-5
Incubation Period (hour) 0
1
2
5.826 ± 0.002 6.019 ± 0.160 7.064 ± 0.005
3.587 ± 0.090 4.208 ± 0.121 8.017 ± 0.487
5.008 ± 0.007 6.386 ± 0.057 12.244 ± 0.247
Growth enhancement of S. au reus
343
which was also a nutritionally limited medium for mammalian cell cultures, was used because, unlike M199, the medium did not contain ATP as a com ponent interfering with bacterial ATP determination. There was no great dif ference in relative light units (RLUs) between the three media before addition of the cells, which were unsupplemented or supplemented with BSA or the extracellular products, therefore none of these media contained a detectable ATP. The presence of extracellular products in the medium (50 ~g/ml) in creased the RLU by about 2.4 times as compared with the nonsupplemented medium after 2 h of incubation. This growth enhancement was roughly 1.9 times as compared to the medium supplemented with BSA. In RPMI 1640 or RPMI 1640 with BSA, a transitory drop of the RLU was observed after 1 h of incubation; it was not observed in the medium with the products. In these and other similar experiments, the staphylococcal growth was in itiated without lag phase due to the presence of the extracellular products (Fig. 1 and Table 1), though the division time was slightly increased compared to growth in nonsupplemented medium (Fig. 1). It was likely that the growth enhancement effect from extracellular products could be attributed to abol ishing of the initial lag phase. The duration of the lag phase in batch cultures is usually inversely related to the size of the cell inoculum and an inoculum dependent lag in several bacteria is reduced by adding supernatant from cul ture of the same organisms. For instance, a lag phase in Nitrosomonas euro pea culture was reduced by the addition of N-acyl homo serine lactone, which has been identified in several gram-negative bacteria as chemical communica tion signals for various physiological processes (2, 5, 12, 16). An anionic com pound of Francisella tularensis and a siderophore of Bacillus also reduced their lag phase by promoting the initiation of cell division (3, 8, 13). Recently, it has been demonstrated in S. aureus that the synthesis of viru lence factors is stimulated with an octapeptide produced and secreted by this organism (1, 9). This peptide seems to activate secretory protein genes and repress surface protein genes by activating the transcription of RNA III which is the intercellular effector of exoprotein response (14, 17). However, it is un known whether this autocrine factor with a molecular weight of 960 Da also possesses growth-stimulating ability. The possible role of this small peptide which accumulates within the midexponential phase in culture is important in the postexponential phase. An RNA III consequently represses surface protein genes (14, 17). The inducible production of secretory proteins by this factor seems to be in conflict with effects on cell division. In conclusion, our results suggest that the culture fluids contains autocrine like factor(s) produced by S. aureus Cowan I strain which stimulate its own growth. In this experiment, a dialysis membrane which retained materials larg er than 1 kDa was used for the preparation of the extracellular products, and an ultrafilter with 5 kDa molecular cutoff also retained the activity in another experiment (data not shown). This may suggest that the factor included in the product preparation possesses a molecular weight of at least 5 kDa. The iden tification of the factor is still left to further experiments, including discrimina-
344
N. Sakata, Y. Usui, and T. Kogure
tion from the known factors mentioned above, but this is a first report which suggests the existence of an autocrine-like growth factor in S. aureus. Acknowledgement. We would like to thank Dr. Torkel Wadstrom for helpful com ments.
References 1. Balaban, N. and R. P. Novick: Autocrine regulation of toxin synthesis by Staphy lococcus aureus. Proc. Natl. Acad. Sci. USA 92 (1995) 1619-1623 2. Beppu, T.: Signal transduction and secondary metabolism: prospects for control ling productivity. Trends Biotechnol. 13 (1995) 264-269 3. Byers, B. R., M. V. Powell, and C. E. Lankford: Iron-chelating hydroxamic acid (schizokinen) activates initiation of cell division in Bacillus megaterium. ]. Bac teriol. 93 (1967) 286-294 4. Carrell, D. T., M. E. Hammond, and W. D. Odell: Evidence for an autocrine/para crine function of chorionic gonadotropin in Xanthomonas maltophilia. Endocri nology 132 (1993) 1085-1089 5. Fuqua, W. c., S. C. Winans, and E. P. Greenberg: Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators.]. Bacteriol. 176(1994)269-275 6. Grover, S., S. R. Woodward, and W. D. Odell: Complete sequence of the gene en coding a chorionic gonadotropin-like protein form Xanthomonas maltophilia. Gene 156 (1995) 75-78 7. Halmann, M., M. Benedict, and]. Mager: Nutritional requirements of Pasteurella tularensis for growth from small inocula.]. Gen. Microbiol. 49 (1967) 451-460 8. Halmann, M. and J. Mager: An endogenously produced substance essential for growth initiation of Pasteurella tularensis.]. Gen. Microbiol. 49 (1967) 461-468 9. Ji, G., R. C. Beavis, and R. P. Novick: Cell density control of staphylococcal viru lence mediated by an octapeptide pheromone. Proc. Natl. Acad. Sci. USA 92 (1995) 12055-12059 10. Kaiser, D. and R. Losick: How and why bacteria talk to each other. Cell 73 (1993) 873-885 11. Kaprelyants, A. S., G. V. Mukamolova, and D. B. Kell: Estimation of dormant Micrococcus luteus cells by penicillin lysis and by resuscitation in cell-free spent culture medium at high dilution. FEMS Microbiol. Lett. 115 (1994) 347-352 12. Kaprelyants, A. S. and D. B. Kell: Do bacteria need to communicate with each other for growth. Trends Microbiol. 4 (1996) 237-242 13. Lankford, C. E., J. R. Walker, ]. B. Reeves, N. H. Nabbut, B. R. Byers, and R.]. Jones: Inoculum-dependent division lag of Bacillus cultures and its relation to an endogenous factor(s) ("schizokinen"). J. Bacteriol. 91 (1996) 1070-1079 14. Novick, R. P., H. F. Ross, S. J. Projan, J. Kornblum, B. Kreiswirth, and S. Mogha zeh: Synthesis of staphylococcal virulence factors is controlled by a regulatory RNA molecule. EMBO].12 (1993) 3967-3975 15. Stanley, P. E.: A concise beginner's guide to rapid microbiology using adenosine tri phosphate (ATP) and luminescence. In: ATP luminescence: rapid methods in microbiology (P. E. Stanley, B.J. McCarthy, and R. Smither, eds.), pp. 1-10. Black well Scientific, Oxford (1989)
Growth enhancement of S. aureus
345
16. Swift, S., N. J. Bainton, and M. K. Winson: Gram-negative bacterial communica tion by N-acyl homo serine lactones: a universal language. Trends Microbiol. 2 (1994) 193-198 17. Vandenesch, F., J. Kornblum, and R. P. Novick: A temporal signal, independent of agr, is required for hla but not spa transcription in Staphylococcus aureus. J. Bac teriol.173 (1991) 6313-6320 Corresponding author: Dr. Noriaki Sakata, Department of Legal Medicine, St. Marianna University School of Medicine, Sugao 2-16-1, Miyamae-ku, Kawasaki 216-8511, Japan, Tel.: ++81449778111 ext. 3556; Fax: ++8144977 3902
Zent.bl. Bakteriol. 289, 339-345 (1999) © Urban & Fischer Verlag http://www.urbanfischer.de/j ournals/ zblbakteriol
Extracellular Product(s) of Staphylococcus aureus Stimulate Their Own Growth Noriaki Sakata\ Yukio Usui h
,
and Tadahisa Kogure 1
Department of Legal Medicine and Department of Microbiology, St. Marianna University School of Medicine, Kawasaki 216-8511, Japan * Present address; Laboratory of Nutrition and Biochemistry, Aoba-Gakuen Junior College, Tokyo
1
2
Received October 5,1998 . Revision received February 10, 1999 . Accepted February 26, 1999
Summary The effect of extracellular products obtained from culture supernatant of Staphylococ cus aureus strain Cowan I on the bacterial growth was studied in a synthetic medium. Addition of the extracellular products to a fresh medium stimulated growth already after 2 h of incubation, with an approximately two-fold increase in cell density as com pared to an un supplemented medium, probably by promoting an initiation of growth accompanied by a reduction of the initial lag phase. The growth-stimulating effect was also monitored as an increase of adenosine triphosphate (ATP) in the bacterial culture during the different phases of growth.
Introduction Many kinds of cellular factors which regulate various cell functions and main tain cooperation between cells in various tissues are well designed in higher multicellular organisms. Even in prokaryotic organisms such as bacteria, intercellular signals seem to mediate cell-cell communication (10). For in stance, several intercellular signals are involved in cell differentiation and morphogenesis, such as fruiting body formation in Myxococcus, erection of aerial hyphae in Streptomyces and the sporulation process in Bacillus (10). Similar systems which are involved in growth have also been suggested to exist 0934-8840/99/289/3-339 $12.00/0
340
N. Sakata, Y. Usui, and T. Kogure
in Francisella (Pasteurella) tularensis, Micrococcus luteus and Xanthomonas maltophilia (4, 6, 7, 8, 11, 12). Considering these examples, such systems of growth regulation through intercellular signals seem to be universal also in the bacterial world. Staphylococcus aureus is a major human pathogen causing a variety of dis eases. This gram-positive coccus can rapidly develop resistance to a variety of antibiotics. Provided such a self-promoting system of cell growth functions also in this bacterial pathogen, new non-antibiotic strategies to prevent and break these infections may be developed. Therefore, we examined a possibil ity that S. aureus produced an autocrine-like factor which was involved in growth regulation.
Materials and Methods Media
Medium 199 (MI99) and RPMI Medium 1640 (RPMI 1640) were purchased from GIBCO BRL, Life Technologies, Inc. (Rockville MD, USA). Brain heart infusion agar (BHI agar) was purchased from Difco Laboratories (Detroit, MI, USA). Culture conditions and preparation of S. aureus extracellular products
One loopful (1 fll) of S. au reus strain Cowan I on BHI agar was inoculated into 10 ml of M199 medium and cultured for 3 h at 37°C. The culture was inoculated into 400 ml of the liquid M199 medium and continued to culture for an additional 18 hat 37°C. The supernatant was obtained by centrifugation (11 000 xg for 20 min at 4°C) and stored at -80°C after filtration by 0.22 flm membrane filter (Millipore corpora tion, Bedford, MA, USA). The stored supernatant was dialyzed against distilled water by using a membrane with 1 kDa of molecular weigth cutoff (Spectrum, Houston, TX, USA) to eliminate the components included in the medium, and then lyophilized. The residue was dissolved in phosphate-buffered saline (PBS) and stored at -20°C. Determination of bacterial cell numbers
Separate standard curves for liquid media were utilized as a reference to determine bac terial cell numbers; the absorbance at 420 nm of a diluted bacterial suspension in the M199 and RPMI 1640 media with 0.1 volume of 1M Tris-HCI, pH 7.5, was measured and duplicate aliquots were plated on BHI agar plates to count bacterial colonies after 24 h incubation at 37°C. Effect of the extracellular products on bacterial growth
One loopful (1 fll) of S. au reus strain Cowan I on BHI agar was grown in 20 ml of M199 for 16 h at 3rC and the cells were washed five times with 40 ml of PBS. The washed cells were cultured, with a density of 3 X 106 cells/ml, at 3rC in M199 sup plemented with 50 flg of protein, or unsupplemented with the extracellular products.
After the specified period of incubation, 1 ml aliquots of culture were mixed with 100 fll of 1M Tris-HCI, pH 7.5, and the absorbance at 420 nm was measured by using M199 with 0.1 volume of 1M Tris-HCl, pH 7.5, as a reference standard.
Growth enhancement of S. aureus
341
Measurement of bacterial ATP during growth A luciferin-Iuciferase assay to determine bacterial ATP was performed in a LUMI COUNTER 700 (MICROTEC NITI-ON, Funabashi, Chiba, Japan) using the follow ing reagents: ATP releasing reagent (TURNER DESIGNS, Mountainview, CA, USA), luciferin-Iuciferase solution (MICROTEC NITI-ON) and HEPES buffer (MICROTEC NITI-ON). Briefly, the bacterial culture was grown overnight in the M199 medium at 37°C and cells were washed five times with PBS. The cells (3 X 10 7 ) were cultured at 37°C in 1.5 ml of RPMI 1640 supplemented with 50 fl.g of protein per ml, or unsup plemented with the extracellular product. 100 fl.l HEPES buffer, was added to a plas tic tube (MICROTEC NITI-ON) including 100 fl.1 of a bacterial culture after different periods of incubation and the mixtures were further incubated with 100 fl.1 of the ATP releasing reagent for 10 seconds. After incubation, 100 fl.l of luciferin-Iuciferase solu tion was added to the mixture and luminescence was measured for 10 seconds. Protein assay A protein assay was performed with BCA protein assay reagents (PIERCE, Rockford, IL, USA), using BSA as a standard.
Results and Discussion In this experiment, an enriched complex culture medium such as BHI seemed to be unsuitable for isolating extracellular products of S. au reus and investi gating their effects because it might contain various nutritional factors which masked the effects of the bacterial products by promoting or repressing the bacterial growth. Therefore, a synthetic medium was used in this study. Espe cially, the M199 medium, originally used for culturing mammalian cells, was considered to be expedient because it contained several vitamin essential for staphylococci, e. g. folic acid and p-aminobenzoic acid, besides amino acids and glucose. S. aureus strain Cowan I grew satisfactorily in this medium (data not shown) and bacterial culture supernatants were obtained (see Materials and Methods). The protein concentration of the culture supernatant was about 30 flg per m!. The effect of the extracellular products on bacterial growth was investigated by culturing S. aureus strain Cowan I in this medium supplemented with the products (Fig. 1). Preparation of bacterial cells for this experiment was also performed by using the M199 medium to eliminate a contamination by exo geneous substances from the culture medium. When staphylococci were incu bated with the extracellular products (50 flg of protein per ml) the cell density was enhanced 2.S-fold after 2 h of incubation, as compared to an unsupple mented medium. The difference in cell density was still maintained after 4 h of incubation. A medium supplemented with bovine serum albumin (BSA) was used as a control to explore the possibility that the presence of proteins might nonspecific ally affect bacterial growth. Addition of BSA (50 flg/ml) seemed to slightly enhance the cell numbers up to 2 h of incubation. However, this effect
342
N. Sakata, Y. Usui, and T. Kogure
of BSA was faint and cell numbers of the 3 h culture were almost equal to those of unsupplemented M199. Living bacterial cells contained ATP levels which were in relation to the cell mass (15). A growth enhancement as described above was confirmed by meas uring total ATP in the culture during bacterial growth (TableI). RPMI 1640,
E ~
Q) ~
Qj
.0
E
10 7
::J
C
iii
'55
'0 III m
10 6
0
4
23 Incubation period (h)
Fig. 1. Growth of S. aureus strain Cowan I in medium supplemented or non supplemented with the extracellular products. The bacterial cells were inoculated into the M199 medium supplemented (closed circle) or non-supplemented (open circle) with the bacterial extracellular products (50 [.tg of proteins/ml), with a density of 3 X 106 cells/ml. The medium supplemented with BSA (50 [.tg/ml open triangle) was also used as a control. The bacterial numbers were determined as described in Materials and Methods. Values are means of duplicate determinations.
Table 1. Total amount of ATP in bacterial culture during growth Relative Light Units (RLU)
Medium Without Cells RPMI1640 0.018 RPMI1640+BSA 0.020 RPMI 1640 + Products 0.026
X
10-5
Incubation Period (hour) 0
1
2
5.826 ± 0.002 6.019 ± 0.160 7.064 ± 0.005
3.587 ± 0.090 4.208 ± 0.121 8.017 ± 0.487
5.008 ± 0.007 6.386 ± 0.057 12.244 ± 0.247
Growth enhancement of S. au reus
343
which was also a nutritionally limited medium for mammalian cell cultures, was used because, unlike M199, the medium did not contain ATP as a com ponent interfering with bacterial ATP determination. There was no great dif ference in relative light units (RLUs) between the three media before addition of the cells, which were unsupplemented or supplemented with BSA or the extracellular products, therefore none of these media contained a detectable ATP. The presence of extracellular products in the medium (50 ~g/ml) in creased the RLU by about 2.4 times as compared with the nonsupplemented medium after 2 h of incubation. This growth enhancement was roughly 1.9 times as compared to the medium supplemented with BSA. In RPMI 1640 or RPMI 1640 with BSA, a transitory drop of the RLU was observed after 1 h of incubation; it was not observed in the medium with the products. In these and other similar experiments, the staphylococcal growth was in itiated without lag phase due to the presence of the extracellular products (Fig. 1 and Table 1), though the division time was slightly increased compared to growth in nonsupplemented medium (Fig. 1). It was likely that the growth enhancement effect from extracellular products could be attributed to abol ishing of the initial lag phase. The duration of the lag phase in batch cultures is usually inversely related to the size of the cell inoculum and an inoculum dependent lag in several bacteria is reduced by adding supernatant from cul ture of the same organisms. For instance, a lag phase in Nitrosomonas euro pea culture was reduced by the addition of N-acyl homo serine lactone, which has been identified in several gram-negative bacteria as chemical communica tion signals for various physiological processes (2, 5, 12, 16). An anionic com pound of Francisella tularensis and a siderophore of Bacillus also reduced their lag phase by promoting the initiation of cell division (3, 8, 13). Recently, it has been demonstrated in S. aureus that the synthesis of viru lence factors is stimulated with an octapeptide produced and secreted by this organism (1, 9). This peptide seems to activate secretory protein genes and repress surface protein genes by activating the transcription of RNA III which is the intercellular effector of exoprotein response (14, 17). However, it is un known whether this autocrine factor with a molecular weight of 960 Da also possesses growth-stimulating ability. The possible role of this small peptide which accumulates within the midexponential phase in culture is important in the postexponential phase. An RNA III consequently represses surface protein genes (14, 17). The inducible production of secretory proteins by this factor seems to be in conflict with effects on cell division. In conclusion, our results suggest that the culture fluids contains autocrine like factor(s) produced by S. aureus Cowan I strain which stimulate its own growth. In this experiment, a dialysis membrane which retained materials larg er than 1 kDa was used for the preparation of the extracellular products, and an ultrafilter with 5 kDa molecular cutoff also retained the activity in another experiment (data not shown). This may suggest that the factor included in the product preparation possesses a molecular weight of at least 5 kDa. The iden tification of the factor is still left to further experiments, including discrimina-
344
N. Sakata, Y. Usui, and T. Kogure
tion from the known factors mentioned above, but this is a first report which suggests the existence of an autocrine-like growth factor in S. aureus. Acknowledgement. We would like to thank Dr. Torkel Wadstrom for helpful com ments.
References 1. Balaban, N. and R. P. Novick: Autocrine regulation of toxin synthesis by Staphy lococcus aureus. Proc. Natl. Acad. Sci. USA 92 (1995) 1619-1623 2. Beppu, T.: Signal transduction and secondary metabolism: prospects for control ling productivity. Trends Biotechnol. 13 (1995) 264-269 3. Byers, B. R., M. V. Powell, and C. E. Lankford: Iron-chelating hydroxamic acid (schizokinen) activates initiation of cell division in Bacillus megaterium. ]. Bac teriol. 93 (1967) 286-294 4. Carrell, D. T., M. E. Hammond, and W. D. Odell: Evidence for an autocrine/para crine function of chorionic gonadotropin in Xanthomonas maltophilia. Endocri nology 132 (1993) 1085-1089 5. Fuqua, W. c., S. C. Winans, and E. P. Greenberg: Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators.]. Bacteriol. 176(1994)269-275 6. Grover, S., S. R. Woodward, and W. D. Odell: Complete sequence of the gene en coding a chorionic gonadotropin-like protein form Xanthomonas maltophilia. Gene 156 (1995) 75-78 7. Halmann, M., M. Benedict, and]. Mager: Nutritional requirements of Pasteurella tularensis for growth from small inocula.]. Gen. Microbiol. 49 (1967) 451-460 8. Halmann, M. and J. Mager: An endogenously produced substance essential for growth initiation of Pasteurella tularensis.]. Gen. Microbiol. 49 (1967) 461-468 9. Ji, G., R. C. Beavis, and R. P. Novick: Cell density control of staphylococcal viru lence mediated by an octapeptide pheromone. Proc. Natl. Acad. Sci. USA 92 (1995) 12055-12059 10. Kaiser, D. and R. Losick: How and why bacteria talk to each other. Cell 73 (1993) 873-885 11. Kaprelyants, A. S., G. V. Mukamolova, and D. B. Kell: Estimation of dormant Micrococcus luteus cells by penicillin lysis and by resuscitation in cell-free spent culture medium at high dilution. FEMS Microbiol. Lett. 115 (1994) 347-352 12. Kaprelyants, A. S. and D. B. Kell: Do bacteria need to communicate with each other for growth. Trends Microbiol. 4 (1996) 237-242 13. Lankford, C. E., J. R. Walker, ]. B. Reeves, N. H. Nabbut, B. R. Byers, and R.]. Jones: Inoculum-dependent division lag of Bacillus cultures and its relation to an endogenous factor(s) ("schizokinen"). J. Bacteriol. 91 (1996) 1070-1079 14. Novick, R. P., H. F. Ross, S. J. Projan, J. Kornblum, B. Kreiswirth, and S. Mogha zeh: Synthesis of staphylococcal virulence factors is controlled by a regulatory RNA molecule. EMBO].12 (1993) 3967-3975 15. Stanley, P. E.: A concise beginner's guide to rapid microbiology using adenosine tri phosphate (ATP) and luminescence. In: ATP luminescence: rapid methods in microbiology (P. E. Stanley, B.J. McCarthy, and R. Smither, eds.), pp. 1-10. Black well Scientific, Oxford (1989)
Growth enhancement of S. aureus
345
16. Swift, S., N. J. Bainton, and M. K. Winson: Gram-negative bacterial communica tion by N-acyl homo serine lactones: a universal language. Trends Microbiol. 2 (1994) 193-198 17. Vandenesch, F., J. Kornblum, and R. P. Novick: A temporal signal, independent of agr, is required for hla but not spa transcription in Staphylococcus aureus. J. Bac teriol.173 (1991) 6313-6320 Corresponding author: Dr. Noriaki Sakata, Department of Legal Medicine, St. Marianna University School of Medicine, Sugao 2-16-1, Miyamae-ku, Kawasaki 216-8511, Japan, Tel.: ++81449778111 ext. 3556; Fax: ++8144977 3902