Feb 26, 1987 - WT-46. United States. C. A. TC. WT-51. United States. C. A. A3d. WT-59. United States. C. A. A8a. MN-036. Japan. C. A. TT. MN-037. Japan. C.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY. Aug. 1987. p. 1983-1986 0099-2240/87/081983-04$02.00/0
Vol. 53. No. 8
Copyright © 1987. American Society for Microbiology
Glucose-6-Phosphate Dehydrogenase Alloenzymes and Their Relationship to Pigmentation in Serratia marcescens DOMINGO GARGALLO. JOSE G. LOREN. JESUS GUINEA. AND MIQUEL VINAS* Laboratoi
of
MicI-obiologv, F(aulty
of
Pharimacy. University o' Barcelona, 08028 Barc(elona,
Received 26 February 1987/Accepted
28
Spaiin
May 1987
A comparative study of environmental and clinical isolates of Serratia marcescens was undertaken with regard to glucose-6-phosphate dehydrogenase (G6PD) electrophoretic mobility and the production of prodigiosin. Two electromorphs of G6PD with electrophoretic mobilities of 0.22 and 0.30 were detected. G6PD electrophoretic type showed a good correlation with the ability to produce prodigiosin.
In the past few years Ser-atia inarcescens has become recognized as an important opportunistic pathogen. The genus Serr-atiai is characterized by the ability to produce a bloodred pigment, prodigiosin. Other procaryotes also produce prodigiosin or prodigiosinlike pigments or both (17). The chemical structure, culture conditions affecting prodigiosin production, and prodigiosin function have been extensively studied (16, 17). However, strains of Serratiti spp. (particularly S. /narces(ens) isolated from adult patients are usually unable to produce prodigiosin. Studies have been done on the relationship between the failure to produce pigment and the pathogenicity of such strains (5. 6. 10). These white strains do not respond in a fashion similar to that of known mutants blocked in prodigiosin biosynthesis on the basis of syntrophic pigmentation (6). According to Holland and Dale (9). plasmids may interfere with pigmentation. either by affecting prodigiosin production or by altering the structure of the bacterial envelope. It has been demonstrated that white Seerr-atia strains are better plasmid recipients than are wild-type pigmented organisms (11). Electrophoretic separation of bacterial proteins is a highly sensitive technique that gives evidence of the similarity and differences between bacterial strains. This technique. with some variations, has been applied in the past few years in taxonomic, as well as genetic, population studies (1, 4. 13. 14). Glucose-6-phosphate dehydrogenase (G6PD) plays a key role in the oxidative branch of the pentose phosphate pathway. However, in Eschreichia coli five electromorphs have been described, proving that they are selectively neutral under standardized conditions (7). G6PD might be used as a genetic marker for evolutionary and taxonomic studies (3). We determined the electrophoretic mobilities of G6PD. prodigiosin production ability. plasmid profiles, and biotyping of a collection of S. inaicescesis from different origins and sources to study the subspecific structure of clinical S. mainrcescens populations. A total of 98 S. nar'cesceiis strains were studied, including ATCC 274. The origins, sources, and relevant characteristics of the strains are shown in Table 1. For biotyping. the strains were tested for their ability to produce prodigiosin by plating them on peptone-glycerol agar (17). Tetrathionate reduction was determined in liquid medium (12) after 2 days of incubation. Carbon source utilization tests were done in *
medium M70 (15): carbohydrates were added at a final concentration of 0.2% (wt/vol). Growth was monitored every day for up to 16 days. Plates of M70 medium without carbon were used as negative controls. Seven carbon sources were tested: ini-erythritol, trigonelline. 4-hydroxybenzoate. 3-hydroxybenzoate. benzoate, DL-carnitine, and lactose. Biotyping was done by the scheme of Grimont and Grimont (8). Protein extracts were prepared from cultures in Trypticase soy broth (BBL Microbiology Systems). After overnight growth of the cultures at 30°C in a rotary shaker. 1-ml samples were used to inoculate 50 ml of prewarmed Trypticase soy broth in 250-ml Erlenmeyer flasks. Growth was monitored spectrophotometrically in a Uvikon 810 (Kontron) spectrophotometer at 600 nm. Bacteria were harvested by centrifugation when the late logarithmic phase was reached and washed by centrifugation and suspension in Tris-citrate buffer (134 mM: pH 7.0). The bacteria were broken by sonication, and each extract was centrifuged. The supernatants were distributed in Eppendorf tubes at 1 ml per tube and stored at -20°C or used immediately for electrophoresis. Vertical polyacrylamide gel electrophoresis was used with discontinuous acrylamide gels. The stacking gel was prepared in Tris hydrochloride (pH 6.8) buffer with 5% acrylamide. and the resolving gel was prepared in Tris hydrochloride (pH 8.8) with 8% acrylamide. The running buffer was Tris-glycine (pH 8.3). Electrophoresis was performed for 20 h at 75 V. The acrylamide gels were transferred to cuvettes and stained for G6PD activity. The histochemical method for G6PD staining was described by Baptist et al. (1). Plasmid presence was tested by the technique of Birnboim and Doly (2). DNA of bacteriophage lambda digested by HiidIII was used as a marker. Of the 98 strains included in this study. 21 produced prodigiosin. According to Ding and Williams (6). S. mnarces(enis strains isolated from adult patients usually do not produce prodigiosin. Two electromorphs of G6PD were detected, with electrophoretic mobilities (0n,1s) of 0.22 and 0.30 (Fig. 1). All pigmented strains plus strains GP and WT-18 had a G6PD with an of 0.30. Strain GP is a nonchromogenic mutant derived from ATCC 274. which belongs to the A2a chromogenic biotype. Strain WT-18 is a nonchromogenic strain that belongs to biotype Ala: this biotype is composed of chromogenic strains, although it includes some occasionally nonchromogenic bacteria (8). In both instances the in,
Corresponding author. 1983
1984
NOTES
APPL. ENVIRON. MICROBIOL.
TABLE 1. Characteristics of 98 isolates of S. minucescents Strain Strain
VG-2 VG-3 VG-4 VG-5 VG-6 VG-7 VG-8 VG-9 VG-11 VG-38 WT-5 WT-10 WT-12 WT-16 WT-17 WT-18 WT-21 WT-31 WT-33 WT-43 WT-46 WT-51 WT-59 MN-036 MN-037 MN-036 MN-042 MN-045 MN-047 MN-049 MN-050 MN-051 MN-052 MN-053 MN-055 MN-056 MN-057 MN-058 MN-059 MN-061 HCP-Rl HCP-R2 HCP-127 HCP-316 HCP-476 HCP-541 HCP-925 HCP-926 HCP-A27 HCP-A52 HCP-A375 HCP-A703 HCP-A824 HCP-A913 HCP-A926 HCP-A991 HCP-A1883 HCP-A2887 HCP-A4870 HCP-A4899 HCP-A15165 HSP-107 HSP-211 HSP-257 HSP-474 HSP-480 HSP-786 HSP-1044
Origin
Switzerland Switzerland Switzerland Switzerland Switzerland Switzerland Switzerland Switzerland Switzerland Switzerland United States United States United States United States United States United States United States United States United States United States United States United States United States
Japan Japan Japan Japan Japan Japan Japan Japan Japan Japan Japan Japan Japan Japan Japan Japan Japan Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain
Source" Soui-ce"
C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C
~~~~~ ~~~~~~~~G6PD) ~ ProdligiosinBitp ~~~~~~form" pr-oductionBitp A A B A A A B A A A B A B A B B B B A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A
+
+
±
A8a A8a A2a A8a A3c A8a A2a A3a A3c A8a A2a A4a
+
A2a
+
A4a A6a Ala
+
±
Ala Alb A8a A4a TC
A3d A8a
-
-
-
-
-
TT A3c TT A5 A8a TCT TCT A5 A5 A3d TCT TCT A5 NB' A5 A8a TT TCT A8a A8a A5 TCT A3d A8a TCT NB NB A4d NB TCT A5 TT TT TT TT TT TCT
AMb TC A4a A3c TCT A8a A4a A4a Continued on following page
NOTES
VOL. 53, 1987 T ABLE
Strain
Origin
HSP-1145 HSP-1186 HSP-1190 HSP-1551 HSP-1562 HSP-1563 HSP-1666 HSP-1667 HSP-2106 HSP-A89 HSP-A134 HSP-A409 HSP-A946 HSP-VK55 HSP-VK58 IPB-4752 CY-429 CY-918
Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain
N-Si N-S3 N-S4 N-SIO N-Sll N-28b N-23a N-24a N-27a N-35a ATCC 274
GP('
1985
I-ConliniUod
Source"
GfrPD
prodiction
Biotype
C C C C C C C C C C C C C C C C C C E E E E E E E E E E ATCC
A A A A A A A A A B B B B B B A A A B B B B A A B B B B B B
-
A8a A4a A4a A8a
Spain
-
-
ASa NB A4b A4a A8a
+ +
A2a A2a
+
A6b Ala A2a A2a
+ + +
+ + +
++ + + + +
-
A3a
A8a A8a Alb Alb Alb Alb A5 A4a Ala
A2a Alb Ala A2a A2a
"C. Clinical; E. environmental. A, in,. =- 0.22: B. in,. = 0.30. NB, Nonbiotypeable. " Mutant unable to produce prodigiosin: obtained from ATCC 274.
relationship between the nonchromogenic strain and the chromogenic ones was strong. On the other hand, all other white strains had an electrophoretic form of G6PD with an m, of 0.22. Only 19 strains were found to contain plasmids. All are nonchromogenic strains. This finding is in agreement with the results of other investigators (9, 11). It must be noted that the 77 nonpigmented strains that had the same G6PD allozyme fall into 11 of the 13 nonpigmented biotypes of S. marcescens: A3a, A3c, A3d, A4a, A4b, AS, A8a, A8b, TCT, TT, and TC (Table 1). Our results demonstrate that there was an absolute correlation between chromogenic phenotype and the G6PD electrophoretic type.
The correlation between inability to produce pigment, plasmid presence, and G6PD electrophoretic form (m,, 0.22) can be explained by assuming that clinical populations of S. marcescens derived from a nonpigmented subpopulation of this species that has this form of G6PD. This subpopulation could have an enhanced ability to accept plasmids (11). This characteristic could give some selective advantage to these strains in a hospital environment, and this implies a clonal selection for these clinical populations. Our present knowledge does not permit us to assess this clonal nature, but further studies to establish the genetic structure of clinical white strains of S. mnarcescens are warranted. We thank Walter Traub, Masaru Nasu, and Alexander von Gravenitz for providing bacterial strains. We are indebted to Miquel Regue for helpful discussion and suggestions.
1
2
4
3
5 _
6
7
8
9
10
LITERATURE CITED 1. Baptist, J. N., C. R. Shaw, and M. Mandel. 1969. Zone electrophoresis of enzymes in bacterial taxonomy. J. Bacteriol. 99:
_
mm aw "
a * .,
_
FIG. 1. G6PD electromorphs of S. marcescens strains. Lanes: 1, GP; 2, ATCC 274; 3, CY-429; 4, MN-036; 5, WT-43; 6, WT-21: 7, = WT-18; 8, VG-3; 9, HCP-316; 10, HSP-A134. A, m,. = 0.22; B, in,.
0.30.
180-188. 2. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523. 3. Bowman, J. E., R. R. Brubaker, H. Frischer, and P. E. Carson. 1967. Characterization of enterobacteria by starch-gel electrophoresis of glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase. J. Bacteriol. 94:544-551. 4. Caugant, D. A., K. Bovre, P. Gaustad, K. Bryn, E. Holten, E. A. Hoiby, and L. 0. Froholm. 1986. Multilocus genotypes deter-
1986
5.
6. 7.
8.
9. 10. 11.
APPL. ENVIRON. MICROBIOL.
NOTES
mined by enzyme electrophoresis of Neisseria meningitidis isolated from patients with systemic disease and from healthy carriers. J. Gen. Microbiol. 132:641-652. Clayton, E., and A. von Graevenitz. 1966. Nonpigmented Serratia marcescens. J. Am. Med. Assoc. 197:111-116. Ding, M.-J., and R. P. Williams. 1983. Biosynthesis of prodigiosin by white strains of Serratia marcescens isolated from patients. J. Clin. Microbiol. 17:476-480. Dykhuizen, D. E., J. Framond, and D. L. Hartl. 1984. Selective neutrality of glucose-6-phosphate dehydrogenase allozymes in Escherichia coli. Mol. Biol. Evol. 1:162-170. Grimont, P. A. D., and F. Grimont. 1981. The genus Serratia, p. 1187-1203. In M. P. Starr, H. Stolp, H. G. Truper, A. Balows, and H. G. Schlegel (ed.), The prokaryotes. Springer-Verlag KG, Berlin. Holland, S., and J. W. Dale. 1980. The effect of resistance plasmids on pigmentation of Serratia marcescens. Microbios Lett. 9:85-89. Katz, D. S., and R. J. Sobieski. 1979. Detection of pigment precursors in white clinical strains of Serratia marcescens. J. Clin. Microbiol. 9:301-303. Lannigan, R., and L. E. Bryan. 1980. Increased frequency of
12.
13. 14.
15. 16. 17.
acceptance of plasmid deoxyribonucleic acid by a nonpigmented strain of Serratia maarcescens. Microbios Lett. 13:123-127. Le Minor, L., M. Chippaux, F. Pichinoty, C. Coynault, and M. Piechaud. 1970. Methodes simples permettant de rechercher la tetrathionate-reductase en cultures liquides ou sur colonies isolees. Ann. Inst. Pasteur 119:733-737. Porras, O., D. A. Caugant, T. Lagergard, and C. SvanborgEden. 1986. Application of multilocus enzyme gel electrophoresis to Haemophilius influenzae. Infect. Immun. 53:71-78. Selander, R. K., D. A. Caugant, H. Ochman, J. M. Musser, M. N. Gilmour, and T. S. Whittam. 1986. Methods of multilocus enzyme electrophoresis for bacterial population genetics and systematics. Appl. Environ. Microbiol. 51:873-884. Veron, M. 1975. Nutrition et taxonomie des enterobacteries. I. Methodes d'etude des auxanogrammes. Ann. Microbiol. (Paris) 126A:267-274. Vintas, M., J. G. Loren, and J. Guinea. 1983. Particulate-bound pigment of Serratia marcescens and its association with the cellular envelopes. Microbios Lett. 24:19-26. Williams, R. P., and S. M. H. Qadri. 1980. The pigment of Serratia, p. 31-75. In A. von Graevenitz and S. J. Rubin (ed.), The genus Serratia. CRC Press, Inc., Boca Raton, Fla.
