CURRENTMICROBIOLOGYVol. 25 (1992), pp. 51-55

Current Microbiology 9 Springer-Verlag New York Inc. 1992

Phase Variations in Bifidobacterium animalis B r u n o Biavati, 1 F r a n c a C r o c i a n i , z P a o l a Mattarelli, 1 a n d Vittorio S c a r d o v i 1 ~Institute of Agrarian Microbiology, University of Bologna, Bologna, Italy; and ZInstitute of Normal and Pathological Cytomorphology, CNR, % Institute of Research "Codivilla Putti," Bologna, Italy

A b s t r a c t . S t r a i n s isolated f r o m r a b b i t , c h i c k e n , a n d rat feces a n d f r o m s e w a g e a n d f e r m e n t e d milk p r o d u c t s , all identified as Bifidobacterium animal&, w e r e f o u n d to s h o w p h a s e v a r i a t i o n s in c o l o n y a p p e a r a n c e a n d in c e l l u l a r m o r p h o l o g y . T h e rate of t r a n s i t i o n in a s w i t c h i n g s y s t e m f r o m o p a q u e to t r a n s p a r e n t c o l o n i e s a n d vice v e r s a w a s d e t e r m i n e d . D i f f e r e n c e s i n p r o t e i n c o m p o n e n t s a n d in p e n i c i l l i n - b i n d i n g p r o t e i n s (PBPs) of the cells f r o m d i f f e r e n t c o l o n y t y p e s are s h o w n .

Bifids w e r e p r e v i o u s l y i s o l a t e d f r o m s o m e c o m m e r cial f e r m e n t e d milk p r o d u c t s a n d r e c o g n i z e d , prim a r i l y b y D N A - D N A h y b r i d i z a t i o n , as Bifidobacterium animalis [1]. D u r i n g that w o r k , c o l o n i e s w e r e c o m m o n l y f o u n d to be d i v e r g i n g in a p p e a r a n c e a n d f o r m e d b y cells o f v e r y different m o r p h o l o g y . Repeated subcultures, phosphoketolase enzymatic tests, a n d p r o t e i n e l e c t r o p h o r e s i s e x c l u d e d c o n t a m i n a t i o n [1]. T h e s e u n e x p e c t e d findings [11] p r o m p t e d us to e n l a r g e the s t u d y o f B. animalis b y e x a m i n i n g o t h e r s t r a i n s f r o m o u r c u l t u r e c o l l e c t i o n that w e r e p r e v i o u s l y i s o l a t e d f r o m rat, c h i c k e n , a n d r a b b i t feces a n d s e w a g e . T h e s e s t r a i n s f o r m e d the b a s i s for the first p r o p o s a l of the species [12] a n d w e r e isolated s o m e 20 y e a r s ago, e a c h f r o m different s a m p l e s of feces a n d sewage. H o w e v e r , the s t r a i n s in the c u l t u r e c o l l e c t i o n i s o l a t e d f r o m f e r m e n t e d milk p r o d u c t s w e r e c o n s i d e r e d of c h i c k e n - r a b b i t fecal origin o n the basis o f n a t i v e p r o t e i n e l e c t r o p h o r e t i c p a t t e r n s [8]. B e c a u s e o f their e x t r e m e u n i f o r m i t y , o n l y o n e strain, M240, w a s t a k e n as r e p r e s e n t a t i v e of the c u l t u r e c o l l e c t i o n g r o u p for this i n v e s t i g a t i o n .

Materials and M e t h o d s Organisms and culture conditions. The B. animalis strains used were T6, T27, T98, T160, and T169 from feces of rat; P16, P17, P23 (ATCC 26536), P32, and P45 from feces of chicken; RA12, RA13, RA14, RA15, RA16, RA20, and RA23 from feces of rabbit; F437 and F439 from sewage; M240 from commercial fermented milk product; R 101-8 (ATCC 25527), type strain of the species [10, 12], from feces of rat. The medium used throughout was trypticase-phytone-yeast extract (TPY) [11]; plates and tubes were incubated at 37~176 in anaerobic jars or plastic bags in which anaerobiosis was estab-

lished by means of the reagent mixtures Anaerocult A and P, respectively (E. Merck, Darmstadt).

Colony observation, Scoring plates for different colony types was a difficult task except for some strains from chickens and rabbits in which the different colony morphotypes were striking (Fig. 1, sections A-C). The existence of different colonies and cell morphology patterns and the transition from one pattern to another were confirmed only after innumerable isolations and scoring of populations from single colonies and from artificial mixtures. Particular care was given to the photographic documentation of colony appearances by use of different types of illumination in a Tessovar stereomicroscope (Zeiss); this helped to define the otherwise difficult-to-comprehend terms we used [i.e., transparent (T) and opaque (O) (see Fig. 1, section A)]. Transition rate determination. Attempts to determine the rate of transition or mutation rate (m) were made according to the procedure of Mayer [9] and with the formula m = In 2 (ME -- M1)/ ( N 2 - N1), where M~ and M2 are the number of the minority population (O or T colonies) arising from plates at times 1 and 2, and NI and Nz are the corresponding total viable counts. The large 150-ramgrid-type petri plates were incubated on plastic bags with Anaerocult P (see above). Viable counts were more easily obtained by means of photographic recording. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Cells washed in phosphate-buffered saline (8 mM Na2HPO4, 2 mM KHzPO4, 140 mM NaC1, 3 mM KCI, pH 7.0) were sonicated, and the total extract (heated at 100~ for 5 min in 5% SDS, 5% mercaptoethanol, 15% glycerol in 0.1 MTris-HCl, pH 6.8) was run for 160 mm with the discontinuousbuffer system of Laemmli [6] on 10% polyacrylamide (0.25% bisacrylamide) slabs of 1.5-ram thickness at 70 mA constant current for 7-8 h at 10~176 in Protean II BIO-RAD apparatus; 50/zg of proteins (BIO-RAD protein assay kit) in a 10-/zlsample per lane was used. Bands were stained with Coomassie brilliant blue R-250.

Address reprint requests to: Dr. Vittorio Scardovi, Istituto Microbiologia Agraria, Via Filippo Re 6, 40126 Bologna, Italy.

52

CURRENT MICROBIOLOGY Vol. 25 (1992)

Fig. 1. Section A: strain P23 (ATCC 27536); (1) and (2), T and O colonies; (3) and (4), T colonies with O papillae viewed under different illumination; (5) and (6), ceils from O and T colonies; (7), mixed type from TPY stab. Section B: strain RAI6; (1), O and T colonies; (2) and (3), cells from the same O and T colonies, respectively. Section C: strain M240 (fermented milk products); (1), sectored O and T colonies; (2) and (3), cells from these colonies, respectively. Section D: SDS-PAGE of total extracts from (1), cells from O colonies, and (2), cells from T colonies. Bar in section C (3) represents 10 tzm (the same for all cell photographs).

B. Biavati et al.: Phase Variations

P B P s detection. [Phenyl-4(n)-3H] benzylpenicillin (Code TRK

779, Amersham) at 1.48 MBq, brought to dryness under vacuum on 10-ml three-piece cap polycarbonate bottle, was added with 200/xl of sonicated cellular extract (see above) containing 4 mg total proteins. After storage at 37~ for 60 rain, 50/zg of cold penicillin was added. A fraction of the mixture was heated to 100~ for 5 min in the presence of SDS, mercaptoethanol, and glycerol (see above), and portions (20 /zl) were run on 8-20% linear polyacrylamide gel gradient (1.5-mm thick; 10-mm wells). Centrifugation of the residual mixture was done at 100,000 g for 45 min in a Beckman 65 rotor to separate a particulate fraction and a supernatant, which were processed and run separately to study the PBPs topology. Slabs (see above) were blotted on nitrocellulose (Millipore cat. no. GSWP 304 FO type GS 0.22-~m pore size) with BIO-RAD Trans-Blot Cell at 4~ overnight at 30 V with 25 mM Tris-HC1, pH 8.3,192 mM glycine, and 20% methanol as transfer buffer. Air-dried filters were fluorographed with EN3HANCE spray (NEN) and exposed to Kodak X-OMAT AR film at - 70~ for some days. 14C-Methylated proteins, CFA 625 (Amersham), were used as molecular weight markers. (We prefer to use nitrocellulose blots for fluorography instead of dried slabs; this procedure does not preclude eventual immunological tests on the stored blots.)

Results and Discussion The relevant results of this first experimental approach are documented in Figs. 1 and 2. Most significant is that the transition in phenotype colony is accompanied by a dramatic change in cell morphology and dimensions. The differences in colony phenotypes O and T are striking in the selected strains P23, RA16, and M240, especially when colonies are viewed under different illumination (Fig. 1, section A3 and 4) or when sectored colonies are formed (Fig. 1, section C). (It should be remembered that the strain M240 from fermented milk products showed the chicken-rabbit electrophoresis pattern; see above.) The difference in colony phenotype is accompanied in these three strains by dramatic changes in cell morphology and size; the cells from T colonies are minute and mostly spherical, whereas those from O colonies show species-specific shapes and dimensions [11]. However, this variation pattern is not shared with by other strains from the same origin; most often colonies were observed and recorded (Fig. 2, sections D and E) that differed in optical parameters much less than did the strains of Fig. 1 (i.e., they were generally opaque or "less" opaque). In such cases differences in cell morphology were still observed but were not striking. B. animalis isolated from rat feces, which included the type strain, were characterized by the formation of even more heterogeneous colonies; some differences in cell

53

morphology were also documented (Fig. 2, sections A-C). In the absence of data concerning the conditions (substrate composition, culture history, conditions of growth, etc.) that could influence the frequency of phenotypic variations, we nervertheless tried to quantitate the transition rate T ~ O colony in a derivative (P23/19/9) obtained by clonal selection from the original strain P23. The same T clone was used for SDS-PAGE and for PBPs detection (see below). The value found was 1.24 x 10 -3 per CFU per generation. The reverse O ~ T variation, in the clone P23/1, phenotype O, occurred at nearly 10 -4. Therefore, this high-frequency switching system of B. animalis is a phase system, in keeping with Trfiper and Kramer [16], who stated that the term phase "should be restricted to well-defined stages of naturally occurring alternating variations." The species B. animalis, found so far in the intestine of rabbit, rat, and chicken, should, therefore, be included among the few Gram-positive bacteria in which phase variations are detected [2, 4, 17]. Attempts were made by means of one-dimensional SDS-PAGE to detect eventual differences in protein components of the cells of the different colony phenotypes; however, to obtain sufficiently uniform populations of the types involved in the switching was a very difficult task, especially for the T cells, prone to switch to O type at high frequency. Having observed that, in TPY liquid medium at pH 6.5, cells from T colonies do develop, albeit slowly, keeping their phenotype longer, we finally obtained a sufficient cell crop by use of numerous 100-ml liquid cultures (pH 6.5) inoculated with 10-ml cultures from single colonies each and pooled just at the beginning of their development. Phase purity was judged microscopically. An example of two lanes of SDS-PAGE is reported in Fig. 1: an abundant protein of MW 54,000 (arrow), which can be seen exclusively in T cell cytoplasm, is now being studied immunologically. A protein band of unknown topology with MW close to 200,000 can be seen only in O cells, as well as a large one in the range 97-100 kiloDalton (kDal); this last protein reacts immunologically with a polyclonal antiserum obtained from a cell-wall-related protein of B. globosum (results not given). It cannot be excluded that such a protein can contribute to the phenotype of the O colonies, as suggested for Neisseria gonorrhoeae [18]. The autoradiography reported in Fig. 2 (section F) unequivocally indicates that the cells derived from the T colony of the strain P23 (small dimensions and globular phenotype) possess a PBP of 71 kDal

54

CURRENT MICROBIOLOGYVol. 25 (1992)

Fig. 2. Sections A and B: strains R 101-8 (ATCC 25527) and T98; (1), colonies of different phenotypes; (2), (3), and (4), cells from colonies of increasing opacity. Section C: strain T6; (1), colonies semi-transparent and opaque with regular or irregular borders; (2), (3), and (4), cells from these colonies, respectively. Section D: strain RAI2; (1), semi-transparent and O colonies; (2) and (3), cells from such colonies, respectively. Section E: strain RA23; (1), semi-transparent and O colonies (and sector therefrom); (2) and (3), cells from such colonies, respectively. Section F: derivatives from strain P23 (ATCC 27536); (1), PBPs in cells from T colonies; (2), PBPs in cells from O colonies. Bar in section E(3) represents 10 ~zm (the same for all cell photographs).

55

B. Biavati et al.: Phase Variations

absent or in traces in the sonicated extracts of the normal or large cells (O phenotype). This is an interesting finding because of the already recognized involvement of PBPs in establishing the cellular shape by specific roles played in peptidoglycan biosynthesis mainly in Escherichia coli [7, 14, 15], but also in some Gram-positive bacteria [3, 5, 13]. Understanding the enzymatic activity of this protein and the role played in bifidal cell morphology should be of interest because, as far as we know, this is the first example of a PBP involved in a high-frequency switching system.

10.

ACKNOWLEDGMENTS

11.

Research supported by the National Research Council of Italy, Special project RAISA, Sub-project no. 4 Paper no. 308. The excellent technical assistance of L. Masi-Guiduzzi in the photodocumentation is acknowledged.

12.

Literature Cited 1. Biavati B, Mattarelli P, Crociani F (1992) Identification of bifidobacteria from fermented milk products. Microbiologica 15:7-13 2. Christensen GD, Baddour LM, Madison BM, Parisi JT, Abraham SN, Hasty DL, Lowrance JH, Josephs JA, Simpson WA (1990) Colonial morphology of staphylococci on Memphis agar: phase variation of slime production, resistance to/3lactam antibiotics, and virulence. J Infect Dis 161:1153-1169 3. Fontana R, Cerini R, Longoni P, Grossato A, Canepari P (1983) Identification of a streptococcal penicillin-binding protein that reacts very slowly with penicillin. J Bacteriol 155:1343-1350 4. Glasgow AC, Hughes KT, Simon MT (1989) Bacterial DNA inversion systems. In: Berg DE, Howe MM (eds) Mobile DNA. American Society of Microbiology, Washington, DC, pp 637-659 5. Grossato A, Cheng YR, Tonin E, Jacques P, Fontana R (1986) Purification of Streptococcus faecium penicillin-binding pro-

6.

7.

8.

9.

13.

14.

15. 16.

17.

18.

tein 5, a multifunctional penicillin binding protein. Microbiologica 9:21-28 Laemmli U K (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685 Matsuhashi M, Wachi M, Ishino F (1990) Machinery for cell growth and division: penicillin-binding proteins and other proteins. Res Microbiol 141:89-102 Mattarelli P, Crociani F, Mucci M, Biavati B (1992) Different electrophoretic patterns of cellular soluble proteins in Bifidobacterium animalis. Microbiologica 15:71-74 Mayer LW (1982) Rates of in vitro changes of gonococcal colony opacity phenotypes. Infect Immun 37:481-485 Mitsuoka T (1969) Vergleichende Untersuchungen iJber die Bifidobakterien aus dem Verdauungstrakt von Menschen und Tieren. Zentralbl Bakteriol [Naturwiss] 210:52-64 Scardovi V (1986) Genus Bifidobacterium Orla-Jensen. In: Sneath PHA, Mair NS, Sharpe ME, Holt JG (eds) Bergey's manual of systematic bacteriology, vol 2. Baltimore: Williams and Wilkins, pp 1418-1434 Scardovi V, Trovatelli LD (1974) Bifidobacterium animalis (Mitsuoka) comb. nov. and the "minimum" and "'subtile" groups of new bifidobacteria found in sewage. Int J Syst Bacteriol 24:21-28 Schuster C, Dobrinski B, Hakenbeck R (1990) Unusual septurn formation in Streptococcus pneumoniae mutants with an alteration in the D,D-carboxypeptidase penicillin-binding protein 3. J Bacteriol 172:6499-6505 Spratt BG (1975) Distinct penicillin-binding involved in the division, elongation, and shape ofEscherichia coli K 12. Proc Natl Acad Sci USA 72:2999-3003 Spratt BG (1983) Penicillin-binding proteins and the future of /3-1actams antibiotics. J Gen Microbiol 129:1247-1260 Trtiper HG, Kramer J (1981) Principles of characterization and identification of prokaryotes. In: Starr M, Stolp H, Balows A, Schlegel H (eds) The prokaryotes, vol 1. Berlin: Springer, pp 173-193 Ueda S, Kuramitsu HK (1988) Molecular basis for the spontaneous generation of colonization-defective mutants of Streptococcus mutans. Mol Microbiol 2(1): 135-140 Walstad DL, Guymon LF, Sparling PF (1977) Altered outer membrane protein in different colonial types of Neisseria gonorrhoeae. J Bacteriol 129:1623-1627