The physiological and genetic diversity of bovine Streptococcus bovis strains1

FEMS Microbiology Ecology 35 (2001) 49^56

www.fems-microbiology.org

The physiological and genetic diversity of bovine Streptococcus bovis strains1 Graeme N. Jarvis a , Amina Kurtovic a , Anthony G. Hay a , James B. Russell a

a;b;

*

Section of Microbiology, Cornell University, Wing Hall, Ithaca, NY 14853, USA b Agricultural Research Service, USDA, Ithaca, NY 14853, USA

Received 30 July 2000; received in revised form 20 October 2000; accepted 20 October 2000

Abstract Laboratory Streptococcus bovis strains and isolates obtained from a steer fed increasing amounts of grain had similar growth characteristics, but they differed in their sensitivity to 2-deoxyglucose (2DG), a non-metabolizable glucose analog. The addition of 2DG decreased both growth rate (0.92 þ 0.34 h31 ) and growth yield (ranging from 25 to 63%), but these differences could not be correlated with diet. However, isolates from a steer fed a 90% grain diet were more prone to 2DG-dependent lysis than those from a hay diet (P 6 0.001). All S. bovis laboratory strains and isolates had an identical restriction fragment length polymorphism pattern, when their 16S rDNA was digested with HaeIII and HhaI. However, when genomic BOX elements were amplified, 5^12 bands were observed, and the S. bovis isolates and laboratory strains could be grouped into 13 different BOX types. Strains 26 and 581AXY2 had the same BOX type, but the remaining laboratory strains did not form closely related clusters. Strains JB1 and K27FF4 were most closely related to each other. Most of the fresh isolates (24 out of 30) could be grouped into a single cluster ( s 90% Dice similarity). This cluster contained isolates from all three diets, but it did not have any of the laboratory strains. The majority (90%) of the isolates obtained from the hay-fed steer exhibited the same BOX type. Because more BOX types were observed if grain was added to the diet, it appears that ruminal S. bovis diversity may be a dietdependent phenomenon. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Diversity; Rumen ; 16S rDNA restriction fragment length polymorphism; BOX-PCR ; 2-Deoxyglucose ; Streptococcus bovis

1. Introduction Streptococcus bovis is frequently isolated from the alimentary tract of cattle, sheep and other ruminants [1], and it causes acute indigestion in cattle that are fed an abundance of grain [2]. During the 1980s, many `streptococci' were re-grouped into other genera (e.g. Lactococcus and Enterococcus), based on 16S rRNA sequencing, DNA homology and cell wall characteristics. S. bovis and S. equinus (the predominant streptococcus associated with the horse alimentary tract) were not reclassi¢ed, and they ap-

* Corresponding author. Tel. : +1 (607) 255-4508; Fax: +1 (607) 255-3904; E-mail: [email protected] 1 Mandatory disclaimer : `Proprietary or brand names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product, and exclusion of others that may be suitable.'

peared to represent a `rather heterogeneous group' within the genus Streptococcus [3]. Earlier work indicated that human and bovine S. bovis strains were genetically homologous [4], but more recently Whitehead and Cotta noted that some human and bovine S. bovis strains could be di¡erentiated using 16S rRNA probes [5]. Klieve et al. [6] digested the 16S rDNA of 30 ruminal S. bovis strains to determine restriction fragment length polymorphism (RFLP). Their results indicated that these isolates were genetically `relatively homogeneous'. However, in each RFLP analysis, only a single restriction enzyme was used, and many of the isolates had di¡erent phage susceptibilities [6]. Growth and fermentation studies indicated that bovine S. bovis strains isolated in California, South Africa and Scotland were very similar [7], but transport assays indicated that S. bovis strains JB1 and 581AXY2 were noticeably di¡erent [8]. S. bovis JB1 (isolated in California) grew diauxically on glucose and lactose, and this regulation was mediated by an inducer exclusion mechanism (glucose enzyme II of the phosphotransferase (PTS)). Strain

0168-6496 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 6 4 9 6 ( 0 0 ) 0 0 1 1 0 - 0

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581AXY2 (isolated in Scotland) had a very low a¤nity for glucose, and approximately 10-fold more glucose was needed to exclude lactose. The glucose PTS of S. bovis JB1 recognizes the nonmetabolizable glucose analog, 2-deoxyglucose (2DG), and 2DG promoted the lysis stationary phase JB1 cultures [9]. Studies with mutants indicated that the 2DG-induced lysis was mediated by the incorporation of 2DG into lipoteichoic acid (LTA) and the failure of this modi¢ed LTA to down-regulate autolytic activity [10]. 2DG decreased the maximum growth rate and growth yield of S. bovis JB1, and S. bovis JB1 has a 2DG-6-phosphate phosphatase that can de-energize the cell [11,12]. The following experiments sought to de¢ne more precisely the phenotypic and genetic diversity of S. bovis laboratory strains, and bovine isolates (obtained form a steer) using 16S rDNA PCR-RFLP, BOX-PCR analyses and exposure to 2DG to determine its e¡ect on growth and lysis. 2. Materials and methods 2.1. Bacterial strains and media S. bovis JB1 was isolated from a cow fed alfalfa hay in Davis, CA, USA, strains 21096C and K27FF4 were obtained from Albrecht Kistner (CSIR, Preetoria, South Africa) and 26, 45S1 an 581AXY2 were obtained from Colin Stewart (Rowett Research Institute, Aberdeen, Scotland, UK). S. bovis strains ATCC 33317T , ATCC 15351, and S. equinus strain 9812T were supplied by Terrence Whitehead (USDA, Peoria, IL, USA). Fresh isolates were obtained from a Holstein steer (approximately 330 kg) that was ¢tted with a ruminal cannula (10 cm in diameter) according to surgical procedures approved by the Cornell Institutional Animal Care and Use Committee. The steer was housed in a tie-stall barn and was fed an 95% ad libitum intake, 12 times per day (2-h intervals commencing at 08:00) with a rotary feeder. The steer was fed the dietary treatments for at least 14 days before sampling. The dietary treatments consisted of three combinations of timothy hay and concentrate (10:90, 55:45, 100:0). Feeds were analyzed for crude protein, neutral detergent ¢ber and acid detergent ¢ber by the Northeast DHI (Ithaca, NY, USA). Ruminal contents were squeezed through four layers of cheesecloth and immediately analyzed for pH with a combination electrode. Volatile fatty acids (VFA) and lactate in cell-free samples were measured by high-performance liquid chromatography (Beckman model 334 liquid chromatograph, model 156 refractive index detector, model 421 CRT data controller, CR1A integrator, Bio-Rad HPX-87H organic acid column, 20-ml loop, 0.013 N H2 SO4 , 0.5 ml min31 , 50³C). The isolation medium contained (per liter) 292 mg of

K2 HPO4 , 292 mg of KH2 PO4 , 240 mg of (NH4 )2 SO4 , 480 mg of NaCl, 100 mg of MgSO4 W7H2 O, 64 mg of CaCl2 W2H2 O, 4 g of Na2 CO3 and 0.6 g of cysteine hydrochloride (pH 6.7). The medium was purged with O2 -free carbon dioxide. Glucose (prepared anaerobically as a separate solution) was then added to achieve a ¢nal concentration of 4 mg ml31 . Ruminal £uid was diluted anaerobically (10-fold increments) in triplicate on two di¡erent days (n = 6) and spread onto the surface of isolation agar plates (Coy Anaerobic Glove Box, Ann Arbor, MI, USA). The plates were anaerobically incubated at 39³C for 18 h. Thirty colonies (10 from each dietary treatment taken on three di¡erent sampling days) were obtained from the endpoint dilution (107 ) plates. 2.2. Phenotypic traits and 2DG-induced lysis Isolates were grown anaerobically (39³C) in 18U150mm tubes that were sealed with butyl rubber stoppers in basal broth (isolation medium that was supplemented with 1.0 mg ml31 Trypticase (BBL Microbiology Systems, Cockeysville, MD, USA), 0.5 mg ml31 yeast extract (Difco, Detroit, MI, USA) and 2 mg ml31 glucose). Growth rate, maximal optical density (OD) and lysis were monitored by changes in OD (1-cm light path, 600 nm), and repeated at least twice for each strain and isolate. 2.3. PCR-RFLP analysis of 16S rDNA The isolates were grown in basal broth (10 ml, 2 mg ml31 glucose, 39³C, 24 h, approximately 160 Wg protein ml31 ). A sub-sample (1 ml) was taken, and the DNA harvested by mechanical disruption (45 s, low speed setting) using a multi-channel beadbeater (Biospec Products, Bartlesville, OK, USA) and sterile glass beads (100-Wm diameter, Biospec Products). The 16S rDNA of the isolates was ampli¢ed using 100 ng of chromosomal DNA and 40 nmol of the universal F27 and R1492 primers [13] and Ready-To-Go PCR beads (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The PCR reaction was performed in a Sprint Thermocycler (Hybaid Ltd., Middlesex, UK). The initial temperature was 94³C (5 min), followed by 35 cycles of 94³C for 30 s; 55³C for 1 min; 72³C for 1 min. The PCR product was double digested using HhaI and HaeIII (3 U and 2 U, respectively) according to manufacturer's instructions (New England Biolabs Inc.). The 16 rDNA restriction fragments were then separated on a 3% agarose gel at 60 V and photographed using the Electrophoresis Documentation and Analysis System 120 (Eastman-Kodak Ltd., Rochester, NY, USA). The 16S rDNA RFLP patterns were compared to one another by using Dice similarity coe¤cient analyses and degrees of homology were determined as previously described [14]. In all gels, S. bovis (strain JB1) was included as a reference, and all 16S rDNA RFLP analyses were repeated at least twice for each strain and isolate.

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2.4. BOX-PCR of genomic DNA Growth conditions and DNA extraction techniques were identical to those described above for PCR-RFLP analyses. The BOX-PCR reaction was undertaken using the BOX1A primer [15], Ready-To-Go PCR beads (Amersham Pharmacia Biotech, Piscataway, NJ, USA) in a PTC-200 Thermal Cycler (MJ Research, Incline Village, NV, USA). A modi¢ed version of the method of Versalovic et al. [15] was employed. Brie£y, the operational parameters were : 94³C for 3 min, followed by 30 cycles of 94³C for 45 s, 50³C for 1 min, 65³C for 6 min; and 65³C for 6 min. The PCR reaction products were then run on a 2% agarose gel at 60 V and the BOX-PCR ¢ngerprints photographed using the Electrophoresis Documentation and Analysis System 120 (Eastman-Kodak Ltd.). Using the criteria of Woods et al. [16], BOX-PCR patterns were considered distinct if a two band or greater di¡erence was observed. The distinct BOX-PCR patterns were then compared to one another using Dice similarity coe¤cient analyses and degrees of homology were determined as previously described [14]. The construction of the dendrogram was based upon the weighted average linkage or UPGAMA method as provided by Quantity One Software (Bio-Rad Laboratories, Richmond, CA, USA). In all gels, S. bovis (strain JB1) was included as a reference, and all BOX-PCR analyses were repeated at least twice for each strain and isolate. 2.5. Statistical analysis The e¡ect of 2DG on the growth rates and glucose yields of the isolates and laboratory strains were subjected to a Student's t-test to determine statistical signi¢cance [14]. 3. Results When the hay in the diet was replaced with the grain mixture, the crude protein content remained relatively consistent, but the neutral detergent ¢ber components decreased (Table 1). Increasing the grain content of the ration led to an increase in ruminal fermentation as observed

Fig. 1. The e¡ect of 2DG and glucose (2 mg ml31 each) compared to control (glucose alone, 2 mg ml31 ) on (a) maximum growth rate and (b) maximum OD of S. bovis laboratory strains (a), 90% grain isolates (R), 45% grain isolates (b), and 0% grain isolates (F).

by the higher concentrations of total VFA and lower ruminal pH (126 mM and pH 5.7 versus 78 mM and pH 6.7, respectively). The acetate to propionate ratio decreased when grain was added to the ration. Because the intake was restricted and the animals were gradually adapted to

Table 1 Chemical composition (percentage of DM) of experimental diets and ruminal parameters Dietary componentsa

100% hay, 0% grain

Crude protein Neutral detergent ¢ber Acid detergent ¢ber Ruminal parameters Total VFA (mM) Acetate to propionate pH

12.4 61.3 42.1

13.7 39.4 25.7

14.4 16.9 8.9

78 4.6 6.7

102 3.3 6.3

126 1.9 5.7

a

55% hay, 45% grain

Dietary ingredients were timothy hay, cracked-corn grain, and soybean meal.

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increasing amounts of grain, ruminal pH was never less than 5.6, and the steer never refused to eat. Rapidly growing, lactate producing cultures that were presumptively identi¢ed as S. bovis were obtained from the steer, irrespective of diet. S. bovis numbers tended to be higher if grain was added to the ration, but this di¡erence was less than 10-fold. The laboratory strains of S. bovis (n = 7) and the fresh isolates (n = 30) grew rapidly on glucose, were nearly homolactic, stained Gram-positive, had ovoid-shaped cells, and were lactose- and starch-positive. Nearly all of the strains fermented ra¤nose, the only exceptions were two fresh isolates from the 45% grain, 55% hay diets. The S. equinus type strain (ATCC 9812) was lactose- and raf¢nose-negative, and growth on starch was weak (Table 2).

All of the S. bovis strains and isolates grew rapidly on glucose, and statistical analyses indicated that the maximum growth rates (Fig. 1a) and growth yields (Fig. 1b) of the laboratory strains and fresh isolates were similar (P s 0.05). The addition of 2DG to the growth medium decreased the maximum growth rates (Fig. 1a) and growth yields (Fig. 1b) of all the strains, and once again the laboratory strains and fresh isolates were similar (P s 0.05). Some of the S. bovis isolates had a higher (glucose) lysis percentage than others, and lysis values ranged from 2% to 61% (Fig. 2a). Statistical analyses indicated the isolates obtained from the 100% hay diet were less prone to lysis than isolates obtained from the 90% grain diet (Table 3). 2DG always enhanced lysis, but the r2 (correlation coef¢cient squared) between lysis after growth on glucose and

Table 2 Physiological and phylogenetic data on bovine S. bovis isolates, laboratory strains and S. equinus (ATCC 9812T ) strain Strain 90% grain isolates 3-09 3-10 3-06 3-07 3-08 3-01 3-02 3-03 3-04 3-05 45% grain isolates 5-17 5-02 5-04 5-05 5-06 5-07 5-08 5-11 5-12 5-9 0% grain isolates 7-17 7-02 7-4 7-6 7-14 7-10 7-20 7-21 7-24 7-14(2) Laboratory strains 45S1 K27FF4 26 581AXY2 JB1 ATCC 3317T ATCC 15351 S. equinus (ATCC 9812) a

Starch

Ra¤nose

Lactose

RFLP pattern

BOX-PCR type

+ + + + + + + + + +

+ + + + + + + + + +

+ + + + + + + + + +

A A A A A A A A A A

3 7 3 8 3 3 3 5 5 3

+ + + + + + + + + +

+ + + + + + + + 3 +

+ + + + + + + + + +

A A A A A A A A A A

5 4 5 6 11 5 5 6 5 5

+ + + + + + + + + +

+ + + + + + + + + +

+ + + + + + + + + +

A A A A A A A A A A

3 5 5 5 5 5 5 5 5 5

+ + + + + + + +a

+ + + + + + + 3

+ + + + + + + 3

A A A A A A A A

9 10 2 2 1 8 13 12

Weak growth.

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lysis after growth on glucose and 2DG was only 35% (Fig. 2b). Statistical comparisons, however, indicated that diet could have a marked impact on 2DG-dependent lysis (Table 3), and several comparisons were signi¢cant (0% versus 90% grain, 45% versus 90% grain, and 0% grain versus the laboratory strains). When the 16S rDNA was ampli¢ed and cut with HhaI and HaeIII, the S. bovis isolates, laboratory strains and S. equinus (ATCC 9812) all had the same RFLP pattern (Fig. 3A). When BOX elements within the genome were ampli¢ed, 5^12 bands were observed on agarose gels (Fig. 3B), and Dice similarity coe¤cient analyses indicated that the S. bovis laboratory strains and isolates were heterogeneous (Table 2). The UPGAMA similarity dendrogram of BOXPCR results indicated that strains 26 and 581AXY2 were of the same type, but the remaining laboratory strains did not form closely related clusters (Fig. 4). Strains JB1 and K27FF4 had Dice similarity coe¤cients that were less

Fig. 3. (A) Representative HaeIII and HhaI restriction endonuclease digestion patterns of PCR-ampli¢ed 16S rDNA of S. bovis strains and isolates. Lane/strain or isolate (diet): 1/123-bp ladder, 2/strain JB1, 3/isolate 3-07 (90% grain),4/isolate 5-11 (45% grain), 5/isolate 7-21 (0% grain), 6/S. equinus (ATCC 9812T ), 7/S. bovis (ATCC 33317T ). (B) Composite agarose gel showing 13 di¡erent BOX-PCR types exhibited by S. bovis strains and isolates. Lane/BOX type (strain or isolate): 1/123-bp ladder, 2/BOX type 1 (JB1), 3/BOX type 2 (26), 4/BOX type 3 (3-06), 5/BOX type 4 (5-02), 6/BOX type 5 (5-04), 7/BOX type 6 (5-11), 8/BOX type 8 (3-07), 9/BOX type 9 (45S1), 10/BOX type 10 (K27FF4), 11/negative control, 12/BOX type 7 (3-10), 13/BOX type 11 (5-06), 14/BOX type 12 (S. equinus ATCC 9812T ), 15/BOX type 13 (S. bovis ATCC 15351), 16/BOX type 8 (S. bovis ATCC 33317T ), 17/123-bp ladder.

than 65%, but were more closely grouped to each other than to any of the other isolates or laboratory strains (Fig. 4, Table 2). Most of the fresh isolates (24 out of 30) could be grouped into a single cluster ( s 90% Dice similarity), and this cluster (composed of BOX types 3 and 5) did not have any of the laboratory strains. The remaining isolates (six out of 30) had Dice similarity coe¤cients that were less than 80%, and tended to have unique BOX types (Fig. 4, Table 2). These isolates were sometimes grouped with laboratory strains, but these clusters had only a few members (Fig. 4, Table 2). Fig. 2. The e¡ect of 2DG and glucose (2 mg ml31 each) compared to control (glucose only, 2 mg ml31 ), on (a) lysis after growth of S. bovis laboratory strains (a), 90% grain isolates (R), 45% grain isolates (b), and 0% grain isolates (F). Averaged data for the S. bovis strains and isolates are shown in (b).

4. Discussion S. bovis is a rapidly growing bacterium that has simple

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Fig. 4. Dendrogram of the 13 BOX-PCR types for the S. bovis laboratory strains, and S. bovis isolates obtained from a steer fed increasing amounts of grain in the diet. The BOX elements were ampli¢ed with a BOX1A primer, with the resulting BOX-PCR patterns compared to one another using Dice similarity coe¤cient analyses and degrees of homology. The construction of the dendrogram was based upon the weighted average linkage or UPGAMA method, with BOX type similarity values expressed on a percentage basis. Results for strain and isolate BOX type designation are shown in Table 2.

nutritional requirements [17]. S. bovis can dominate the rumen when large amounts of soluble carbohydrate (sugars or starch) are fed [2], and under these conditions, the success of S. bovis is determined by its ability to `generate more ATP per unit time' than other ruminal bacteria [18]. S. bovis can switch its fermentation from lactate to acetate, formate and ethanol to obtain more ATP, but it is not able to compete as e¡ectively with other ruminal bacteria if carbohydrate is limiting [19]. S. bovis does not have any unique energy sources, but previous work indicated that S. bovis strains could be nonselectively isolated from the rumen so long as the agar plates were composed of a minimal medium, and colonies were picked at 18 h [20]. S. bovis utilizes starch, lactose and ra¤nose, but S. equinus grows poorly on starch, lactose and ra¤nose [1]. All of the laboratory strains and the fresh isolates grew well on starch and lactose and all but two of the fresh isolates utilized ra¤nose as an energy source (Table 2). Based on these results, it appeared that the fresh isolates should be classi¢ed as S. bovis rather than S. equinus, but the independence of these two species is at best tenuous. 16S rDNA sequencing indicated that S. bovis JB1 and S. equinus 9812 were virtually identical (only two out of 356 bases di¡ered, GenBank). Hardie [1] indicated that S. equinus might be nothing

more than a lactose-negative variant of S. bovis, and that di¡erences in starch and ra¤nose utilization are not particularly striking. We only examined a single strain of S. equinus, but this type strain had an 16S rDNA RFLP pattern that was identical to all S. bovis strains and isolates. The S. equinus strain (ATCC 9812T ) had a unique BOX type, but it still grouped in amongst the S. bovis strains and isolates on the dendrogram (Fig. 4). These results support the idea that S. equinus and S. bovis are indeed the same Streptococcus species, as has previously been suggested [3]. All of the laboratory strains of S. bovis and the fresh isolates grew rapidly on glucose (1.71 þ 0.15 h31 ). Previous work with S. bovis JB1 had indicated that the maximum speci¢c growth rate could be as high as 2.0 h31 , but those cultures were repeatedly transferred every 12 h [19]. 2DG decreased the maximum growth rates of all of the S. bovis strains and isolates, but the response was variable (0.92 þ 0.34 h31 ). The laboratory strains of S. bovis and the fresh isolates had similar growth yields (10.6 þ 0.2 g cell protein mmol glucose31 ), and 2DG (2 mg ml31 ) decreased the growth yields of all the isolates (6.4 þ 1.0 g cell protein mmol glucose31 ). However, the range was as great as 25^63%. These results indicated that the S. bovis strains di¡ered in their sensitivity to 2DG, but diet e¡ects could not be demonstrated from growth yield data alone. 15 N studies indicated that microbial protein turnover in the rumen can be as great as 50% [21]. This turnover has often been explained by protozoal predation, but the turnovers of defaunated sheep were nearly as large [22]. This latter result indicated that ruminal bacteria seemed to have high natural lysis rates. S. bovis is a Lance¢eld group D streptococcus that has kojibiose in its LTAs. Kojibiose has an K-1,2 glucose linkage, and this disaccharide is needed for autolytic regulation [10]. 2DG can be incorporated Table 3 Statistical comparisons of natural lysis and 2DG-induced lysis for S. bovis strains isolated from cattle fed di¡erent amounts of grain to create di¡erent ruminal pH Lysis conditiona

Comparison

Signi¢canceb

Glucose Glucose Glucose Glucose Glucose Glucose Glucose Glucose Glucose Glucose Glucose Glucose

0% grain versus 45% grain 45% grain versus 90% grain 0% grain versus 90% grain Lab strains versus 0% grain Lab strains versus 45% grain Lab strains versus 90% grain 0% grain versus 45% grain 45% grain versus 90% grain 0% grain versus 90% grain Lab strains versus 0% grain Lab strains versus 45% grain Lab strains versus 90% grain

NS NS * NS NS NS NS ** *** * NS NS

alone alone alone alone alone alone plus 2DG plus 2DG plus 2DG plus 2DG plus 2DG plus 2DG

a Cultures were incubated for 24 h at 39³C, and lysis was determined from the decrease in OD. Glucose and 2DG were provided at 2 mg ml31 . b Statistical signi¢cance of Student's t-test: *P 6 0.05; **P 6 0.01; ***P 6 0.001; NS, not signi¢cant.

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into the LTAs of S. bovis, but 2DG cannot be linked to glucose to form kojibiose. S. bovis JB1 is a remarkably stable laboratory strain, but previous work indicated that its lysis rate could be greatly enhanced by 2DG. 2DG is rarely found in nature, but it magni¢ed and enhanced the natural lysis rate of our S. bovis isolates. Isolates that were highly sensitive to 2DG also had high natural lysis rates, and diet e¡ects were readily apparent. These results supported the idea that S. bovis isolates were not always physiologically similar and indicated that S. bovis selection in the rumen may be in£uenced by the availability of carbohydrate. When the steer was consuming hay, the rate of carbohydrate fermentation was slow, and the S. bovis isolates had a low rate of 2DGdependent lysis. If grain was added to the diet, fermentation rate increased, pH declined and the S. bovis isolates had a higher rate of 2DG-dependent lysis. Whitehead and Cotta [5] noted that eight bovine strains of S. bovis reacted with a 16S rRNA probe that was designed for S. bovis JB1, but DNA^DNA hybridizations indicated that there were at least two distinct bovine S. bovis groups [23]. When Klieve et al. [6] obtained isolates from cattle fed large amounts of grain or only hay, all of the isolates appeared to be genetically homogeneous. This latter study used only 16S rDNA RFLP patterns to di¡erentiate the strains, and only a single enzyme was used for each digest. When we did double digests (HaeIII and HhaI) our results indicated that our strains and isolates formed a single RFLP pattern, however the converse was true for the BOX-PCR results. Previous work indicated that BOX-PCR was a more sensitive technique for detecting strain di¡erences than 16S rRNA RFLP [15,24], and BOX-PCR analyses indicated that at least 12 di¡erent BOX types of S. bovis were observed in the present study. BOX-PCR is a technique that has several advantages over 16S rDNA RFLP: (1) 16S rDNA only accounts for a small fraction of the total DNA, but many bacteria have BOX elements scattered throughout the entire genome, (2) some parts of the 16S rDNA sequence are variable, but the boxA, boxB and boxC sub-units are highly conserved, (3) even double digests of the 16S rDNA generated less than eight bands, but BOX-PCR of the S. bovis genome generated as many as 12 bands of DNA, and (4) point mutations can destroy or modify restriction sites for 16S rDNA RFLP, but such mutations would have no a¡ect on BOX-PCR [15,24]. The BOX-PCR analyses indicated that the majority of our fresh isolates clustered with the ATCC and other laboratory S. bovis strains. However, both JB1 and K27FF4 formed a distinct cluster ( s 65% similarity, Fig. 4). This result is in agreement with the DNA^DNA hybridization studies of Nelms et al. [23]. Schleifer and Kilpper-Ba«lz [3] concluded that the S. bovis phylogenetic group is composed of several genospecies, but this conclusion was based only on DNA^DNA homology and biochemical characteristics. Our BOX-PCR analyses indicated that

55

the laboratory strains formed seven distinct groups ( s 65% similarity). Most of the fresh isolates could be grouped into a single cluster composed of BOX types 3 and 5, but six other BOX types were also observed. When hay was fed to the animal, 90% of the isolates obtained were BOX type 5, however when grain was introduced into the diet, genetic diversity increased (Table 2). Recent work indicated that Escherichia coli diversity in cattle was overshadowed by animal e¡ects (di¡erences), signi¢cant diet-dependent di¡erences within an animal could not be demonstrated, and physiological variations were not apparent [25]. The present work indicates that S. bovis isolates were physiologically (2DG sensitivities) and genetically (BOX-PCR) diverse. Because isolate diversity from a single animal fed di¡erent rations (hay versus grain) was nearly as great as the diversity of laboratory strains that came from di¡erent animals, and ration e¡ects could be demonstrated, it appears that diet can have an impact on the selection of S. bovis within the rumen microbial ecosystem. However, it should be noted that the present study only compared strains from a single steer (n = 30) with a relatively modest number of laboratory stains (n = 8). Further work using more strains taken from additional animals will be needed to determine which e¡ect (diet or animal) is more important. Acknowledgements J.B.R. is a member of the U.S. Dairy Forage Research Center (Madison, WI, USA). A.K. was the recipient of a scholarship from the Cornell Hughes Undergraduate Research Program.

References [1] Hardie, J.M. (1986) Other streptococci. In: Bergey's Manual of Systematic Bacteriology (Sneath, P.N., Mair, N.S., Sharpe, M.E. and Holt J.G., Eds.), pp. 1068^1071. Williams and Wilkins, Baltimore, MD. [2] Hungate, R.E., Dougherty, R.W., Bryant, M.P. and Cello, R.M. (1952) Microbiological and physiological changes associated with acute indigestion in sheep. Cornell Vet. 42, 423^449. [3] Schleifer, K.H. and Kilpper-Ba«lz, R. (1987) Molecular and chemotaxonomic approaches to the classi¢cation of streptococci, enterococci and lactococci : a review. Syst. Appl. Microbiol. 10, 1^19. [4] Coykendall, A.L. and Gustafson, K.B. (1985) Deoxyribonucleic acid hybridizations among strains of Streptococcus salivarius and Streptococcus bovis. Int. J. Syst. Bacteriol. 35, 274^280. [5] Whitehead, T.R. and Cotta, M.A. (2000) Development of molecular methods for identi¢cation of Streptococcus bovis from human and ruminal origins. FEMS Microbiol. Lett. 182, 237^240. [6] Klieve, A.V., Heck, G.L., Prince, M.A. and Shu, Q. (1999) Genetic homogenity and phage suspectibility of ruminal strains of Streptococcus bovis isolated in Australia. Lett. Appl. Microbiol. 29, 108^112. [7] Russell, J.B. and Robinson, P.H. (1984) Compositions and characteristics of strains of Streptococcus bovis. J. Dairy Sci. 67, 1525^1531. [8] Kearns, D.B. and Russell, J.B. (1996) Catabolite repression in a dia-

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[9]

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G.N. Jarvis et al. / FEMS Microbiology Ecology 35 (2001) 49^56 uxic and non-diauxic strain of Streptococcus bovis. Curr. Microbiol. 32, 216^219. Russell, J.B. and Wells, J.E. (1997) The ability of 2-deoxyglucose to promote the lysis of Streptococcus bovis JB1 via a mechanism involving cell wall stability. Curr. Microbiol. 35, 299^304. Bond, D.R., Tsai, B.M. and Russell, J.B. (1999) Physiological characterization of Streptococcus bovis mutants that can resist 2-deoxyglucose-induced lysis. Microbiology 145, 2977^2985. Cook, G.M., Kearns, D.B., Russell, J.B., Reizer, J. and Saier, J.M.H. (1995) Regulation of the lactose phosphotransferase system of Streptococcus bovis by glucose: independence of inducer exclusion and expulsion mechanisms. Microbiology 141, 2261^2269. Cook, G.M., Ye, J.J., Russell, J.B. and Saier, M.H. (1995) Properties of two sugar phosphate phosphatases from Streptococcus bovis and their involvement in inducer explusion. J. Bacteriol. 177, 7007^7009. Lane, D.J. (1999) 16S/23S rRNA sequencing. In: Nucleic Acid Techniques in Bacteria (Stackebrandt, E. and Goodfellow, M., Eds.), pp. 115^175. Wiley, New York. Sneath, P.H.A. and Sokal, R.R. (1973) Numerical Taxonomy ^ the Principles and Practice of Numerical Classi¢cation. W.H. Freeman and Company, San Francisco, CA. Versalovic, J., Schneider, M., de Bruijn, F.J. and Lupski, J.R. (1994) Genomic ¢ngerprinting of bacteria using repetitive sequence-based polymerase chain reaction. Methods Cell. Mol. Biol. 5, 25^40. Woods, C.R., Versalovic, J., Koeuth, T. and Lupski, J.R. (1992) Analysis of relationships among isolates of Citrobacter diversus by using DNA ¢ngerprints generated by repetitive sequence-based primers in the polymerase chain reaction. J. Clin. Microbiol. 30, 2921^ 2929. Wolin, M.J., Manning, G.B. and Nelson, W.O. (1959) Ammonia salts as a sole source of nitrogen for the growth of Streptococcus bovis. J. Bacteriol. 78, 147^149.

[18] Hungate, R.E. (1979) Evolution of a microbial ecologist. Ann. Rev. Microbiol. 33, 1^20. [19] Russell, J.B. and Baldwin, R.L. (1979) Comparison of maintenance energy expenditures and growth yields among several rumen bacteria grown on continuous culture. Appl. Environ. Microbiol. 37, 537^543. [20] Wells, J.E., Krause, D.O., Callaway, T.R. and Russell, J.B. (1997) A bacteriocin-mediated antagonism by ruminal lactobacilli against Streptococcus bovis. FEMS Microbiol. Ecol. 22, 237^243. [21] Nolan, J.V. (l975) Quantitative models of nitrogen metabolism in sheep. In: Digestion and Metabolism in the Ruminant (MacDonald, I.W. and Warner, A.C.I., Eds.), pp. 416^431. Univ. New England Publishing Unit, Arimdale. [22] Krebs, G.L., Leng, R.A. and Nolan, J.V. (1989) Microbial biomass and production rates in the rumen of faunated and fauna-free sheep on low protein ¢brous feeds with or without nitrogen supplementation. In: The Roles of Protozoa and Fungi in Ruminant Digestion (Nolan, J.V., Leng, R.A. and Demeyer, D.I., Eds.), p. 295. Penambul, Armidale. [23] Nelms, L.F., Odelson, D.A., Whitehead, T.R. and Hespell, R.B. (1995) Di¡erentiation of ruminal and human Streptococcus bovis strains by DNA homology and 16S rRNA probes. Curr. Microbiol. 31, 294^300. [24] Rademaker, J.L.W. and de Bruijn, F.J. (1997) Characterization and classi¢cation of microbes by rep-PCR genomic ¢ngerprinting and computer-assisted pattern analysis. In: DNA Markers : Protocols, Applications and Overviews (Caetano-Anolles, G. and Gressho¡, P.M., Eds.), pp. 151^171. J. Wiley and Sons, New York. [25] Jarvis, G.N., Kizoulis, M.G., Diez-Gonzalez, F. and Russell, J.B. (2000) The genetic diversity of predominant Escherichia coli strains isolated from cattle fed various amounts of hay and grain. FEMS Microbiol. Ecol. 32, 225^233.

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