NOTES. for 12 h, on both rumen fluid medium (11) and. was determined by enzyme assay and by reac- One unit of activity of APase is defined as a

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1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1977, p Vol. 34, No. 5 Copyright 1977 American Society for Microbiology Printed in U.S.A. NOTES Alkaline Phosphatase Activity of Rumen Bacteria K.-J. CHENG'* AND J. W. COSTERTON2 Research Station, Agriculture Canada, Lethbridge, Alberta, TIJ 4B1,I and Department of Biology, University of Calgary, Calgary, Alberta, T2N 1N4,2 Canada Received for publication 15 February 1977 Of the 54 strains of rumen bacteria examined for alkaline phosphatase (APase) production, 9 of 33 gram-negative strains and none of 21 gram-positive strains produced the enzyme. The APase of the cells of the three strains of Bacteroides ruminicola that produced significant amounts of the enzyme was located in the periplasmic area of the cell envelope, whereas the enzyme produced by some strains of Selenomonas ruminantium and Succinivibrio dextrinosolvens was associated with the outer membrane. The localization of APase production in the cells of natural populations of rumen bacteria from hay-fed sheep was accomplished by reaction product deposition, and both the proportion of APaseproducing bacteria and the location of the enzyme in the cell envelope of the producing cells could be determined. We suggest that this procedure is useful in detecting shifts in the bacterial population and the release of cell-bound APase that accompany feedlot bloat and other sequelae of dietary manipulation in ruminants. We have reported that shifting hay-fed cows vol/vol) and incubated further with shaking. All to a high-energy, fine-particle-size concentrate experiments described herein used Scott and diet increases the level of total bacterial alkaline Dehority's synthetic medium with hemin and phosphatase (APase) in rumen fluid 60- to 70-40% rumen fluid, unless otherwise stated. fold, changes a typically periplasmic APase location to a mixed periplasmic and outer cell wall ria from M. P. Bryant's culture collection (De- Forty-nine strains of anaerobic rumen bacte- location, and increases cell-free APase levels partment of Dairy Science, University of Illinois), three strains from G. A. Jones (Depart- about 120-fold (21). We have speculated that these changes reflect both in bacterial cell lysis ment of Dairy and Food Science, University of and the selection of an altered bacterial population that includes more APase producers (21). tion were tested for their capacity to produce Saskatchewan), and two strains from our collec- Prior examination of two common rumen bacteria (Bacteroides ruminicola and B. succino- for 12 h, on both rumen fluid medium (11) and constitutive APase. Stock strains were incubated genes) had shown them to be constitutive producers of a periplasmic APase (19), and we unously (37), and the APase activity of the cultures modified artificial medium as described previdertook this present study to assess APase production by 54 strains of rumen bacteria because tion product deposition (19, 21). was determined by enzyme assay and by reac- APase determinations may be useful for detecting diet-dependent shifts in populations of ru- change of 1 optical density unit at 420 nm/min One unit of activity of APase is defined as a men bacteria. per ml of cultures or rumen fluids, usingp-nitrophenyl-phosphate as substrate, at 250C (19, 21). The anaerobic technique used for culturing the bacteria was essentially that of Hungate (29) Natural populations of rumen bacteria were as modified by Bryant and Burkey (8). Rumen recovered from four sheep fed hay ad lib. Rumen bacteria were cultured anaerobically for 10 h at contents were collected by stomach tube with a 38 C in prereduced rumen fluid medium (11) or hand vacuum pump, filtered through cheesecloth, and analyzed for APase production by Scott and Dehority's synthetic medium (37), modified to contain 5 x 10' M hemin and with reaction product deposition as described previously (21). Fixation, embedding, staining, and 40% rumen fluid, on a rotary shaker (75 rpm). They were then inoculated into 300 ml of fresh electron microscopy procedures were as described previously (2). medium in 500-ml round-bottom flasks (5%, 586

2 VOL. 34, 1977 Nine of the 33 gram-negative strains tested showed a constitutive ability to produce APase (Table 1), whereas none of the 21 gram-positive strains produced the enzyme. Among the gramnegative organisms, three strains (23, B14, and 118B) of B. ruminicola (15) produced large amounts of the enzyme, whereas one strain (GA33) produced none. One strain (S85) of B. succinogenes showed weak enzyme activity, whereas two strains (70 and H18) of B. amylophilus (2, 12), one strain (316) of B. clostridiiformis (18), and an unidentified Bacteroides (17) strain (B85) showed no APase production. Two strains (24 and 22B) of Succinivibrio dextrinosolvens showed strong APase activity, and two strains (HD1 and HD4) of Selenomonas ruminantium (5) showed moderate APase activity, whereas two other strains (PC18 and GA31) produced no APase. Another gram-negative rumen isolate (LMR lancent strain B36) showed weak APase activity. Seven strains (B199, 7, B3C7, 20, D89, B337 and B36) of Ruminococcus albus (16, 17), two strains (D157 and B133) of Ruminococcus sp. (16), four strains (T81, B159, B116, and B178) of Megasphaera elsdenii (17, 36), one strain (40) of Lachnospira multiparus (14), one Borrelia strain (B25) (4), and two B- 385-like (group 10) isolates (B385-1 and 2-33) (6, 13) did not produce APase. The gram-positive isolates that failed to produce APase included four strains (03, D1, 49, and A3m) of Butyrivibrio fibrisolvens (13, 20, 25), four strains (C94, B1C45, FD1, and B146) of TABLE 1. NOTES 587 Ruminococcus flavefaciens (16, 21), two strains (B1C23 and GA195) of Eubacterium ruminatium (6), one strain (PDO 4822) of Clostridium (7), three strains (Pel3, Pel5, and Peli2) of Coprococcus (39), three strains (B62, T185, and GA-1) of Lactobacillus (12, 17), one strain (L34) of Ramibacterium (17), one strain of Streptococcus bovis (22), and one isolate from each of the C-1 and C-3 groups (17). The nine APase-producing strains were examined in pure culture, and it was determined that the enzyme was, produced constitutively (the inorganic phosphorus in the medium was mg/ml) and that the enzyme was essentially cell bound throughout growth in batch culture. The location of the APase produced by eight of these strains was located by reaction product deposition followed by electron microscopy (21, 27) (Table 1). Reaction product deposition of natural bacterial rumen populations showed both the proportion of enzyme-producing cells in the rumen and the location of the enzyme within these cells (Fig. 1). We examined bacterial cells from the rumen of four hay-fed sheep by reaction product deposition and found about 27% to have APase activity. Among these reactive cells from these natural populations, 56% showed a predominantly periplasmic location of APase, 6% showed activity in the periplasm and the outer membrane, and 19% showed activity in the periplasm, the outer membrane, and the capsule. Some cells (13%) showed APase activity associ- Cell-associated APase ofgram-negative rumen bacteria APase activ- Localizationb Organism Strain Reference ity" Outer Permembrane plasm Bacteroides ruminicola Bacteroides ruminicola B Bacteroides ruminicola 118B Bacteroides succinogenes S Selenomonas ruminantium HDlc Selenomonas ruminantium HD4c Succinivibrio dextrinosolvens Succinivibrio dextrinosolvens 22Bd LMR lancent (group 11) B a Units per 30 ml of culture. Optical density units for strains 23, B14, 118B, S85, HD1, HD4, 24, 22B, and B36 were 1.920, 1.936, 2.148, 1.650, 1.885, 1.825, 1.355, 1.258, and 1.356, respectively. Thirty milliliters of 12-h cells of cultures was centrifuged, and supernatant fluids were assayed for cell-free APase activity. The cell pellets obtained were suspended into 10 ml of 0.05 M tris(hydroxymethyl)aminomethane (ph 8.4), and cells were sonified for APase determination. No cell-free APase activity was detected. Results are the average of three experiments. b APase was localized by the deposition of electron-dense lead phosphate after incubation of the cells in a modified reaction mixture as previously described (9, 21). c Originally obtained from Lorraine Gall, National Dairy Research Laboratories, Oakdale, N.Y. d Originally obtained from S. R. Elsden, A. R. C. Unit for Microbiology, University of Sheffield, Sheffield, England; unpublished data of Sheila Wilson and S. R. Elsden. -, APase was not localized.

3 Wi:#s44 Z n X U" I Downloaded from on January 18, 2019 by guest FIG. 1. Electron micrograph of cells from the natural rumen population of hay-fed sheep that were tested for APase localization by reaction product deposition. Note that only some cells are enzyme producers and that the localization of the enzymes varies among the different cells seen here. In some cells (P), the enzyme activity is largely periplasmic; in others (W9, it is associated with the outer membrane of the cell wall; in others, it is associated with both the periplasm and the outer membrane (PW); and in others (PC), it is associated with the periplasm, the outer membrane, and the capsule. Bar = 0.1,um. 588

4 VOL. 34, 1977 ated only with their outer membrane, whereas 6% showed the enzyme activity only in their capsules. Control preparations in which the substrate was omitted showed no electron density in the periplasmic area of any of the bacterial cells, but electron-dense material, which could be confused with reaction product, was seen in association with the outer membrane or the capsule, or both, in a small proportion of the cells. The constitutive ability to produce APase has been used in the identification of staphylococci (1) and in the differentiation of Serratia from Enterobacter (40), and it has been suggested that constitutive APase production is correlated with pathogenicity in staphylococci (35). More recently, Porschen and Spaulding (34) made a valuable survey of anaerobic bacteria of medical importance in which they noted strong APase production by B. ruminicola (three strains) and B. fragilis (27 strains), whereas B. clostridiiformis, B. corradens, B. pneumosintes, and B. melaninogenicus were negative. Among the grampositive bacteria in their study, three of seven species of Peptostreptococcus were positive, as were two of three species of Clostridium. Bray and King (3) concluded that phosphatase production is a valuable taxonomic characteristic in the identification of anaerobic pathogens. The situation seems less clear-cut in the difficult area of rumen bacterial taxonomy because of the striking heterogeneity in APase production within genera and even within species. The heterogeneity may reflect important taxonomic problems in the classification of these organisms, and we are expanding our testing program to address these potential problems. We have used the reaction product deposition technique, which has been recently questioned (38) and more recently vindicated (23, 24, 27, 31-33), to show that the APase of cells in pure cultures may exist in the periplasmic space or in association with the outer membrane and that the APase of cells in natural rumen populations may exist in these same locations, in association with capsular material, or in many combinations of these locations. The enzyme may be lost from these cell envelope locations upon cell lysis, and its release may free it from a somewhat cryptic state in the intact cell envelope (26, 27, 31) so that both extracellular APase and total APase are elevated by the bacterial lysis that accompanies some dietary shifts (21). Another part of an overall increase in the bacterial APase level in the rumen, when cows are changed from a hay to a concentrate diet, involves a shift in which an adapted population of rumen bacteria that includes additional NOTES 589 APase producers is selected (21). Thus it is evident from this and previous studies that bacteria similar to the nine strains of pure cultures that produced APase are usually more numerous in cattle fed a concentrate diet (6, 10, 18, 28, 30). This population shift due to change in diet also produces a lower final ph of fermentation and different fermentation products (essentially propionate) in the rumen (6, 10, 18, 28, 30). Hence, APase can be used as an indicator for the propionate type of fermentation (high-concentrate diet) in contrast to the acetate type of fermentation (high-roughage diet). With reaction product deposition, we can determine the proportion of APase-producing cells in natural bacterial populations in the rumen, and we have shown that 27% of the bacterial cells in the rumens of the four hay-fed sheep used in this study were APase producers. Radical shifts in the balance of bacterial groups within the rumen would be expected to cause a significant shift in the proportion of APase producers, and this balance may be valuable in monitoring the effects of dietary manipulations of the animal on the bacterial population of the rumen. We wish to acknowledge the generous support of the Alberta Agricultural Research Trust and of Agriculture Canada. LITERATURE CITED 1. Barber, M., and S. W. A. Kuper Identification of Staphylococcus pyogenes by the phosphatase reaction. J. Pathol. Bacteriol. 63: Blackburn, T. H., and P. N. Hobson Further studies on the isolation of proteolytic bacteria from the sheep rumen. J. Gen. Microbiol. 29: Bray, J., and E. J. King The phosphatase reaction as an aid to the identification of micro-organisms using phenolphthalein phosphate as substrate. J. Pathol. Bacteriol. 55: Bryant, M. P The isolation and characteristics of a spirochete from the bovine rumen. J. Bacteriol. 64: Bryant, M. P The characteristics of strains of Selenomonas isolated from bovine rumen contents. J. Bacteriol. 72: Bryant, M. P Bacterial species of the rumen. Bacteriol. Rev. 23: Bryant, M. P., B. F. Barrentine, J. F. Sykes, I. M. Robinson, C. V. Shawver, and L. W. Williams Predominant bacteria in the rumen of cattle on bloat-provoking ladino clover pasture. J. Dairy Sci. 43: Bryant, M. P., and L. A. Burkey Cultural methods and some characteristics of some of the more numerous groups of bacteria in the bovine rumen. J. Dairy Sci. 36: Bryant, M. P., and R. N. Doetsch A study of actively cellulolytic rod-shaped bacteria of the bovine rumen. J. Dairy Sci. 37: Bryant, M. P., and I. L. 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5 590 NOTES non-selective culture medium for ruminal bacteria and its use in determining diurnal variation in numbers of bacteria in the rumen. J. Dairy Sci. 44: Bryant, M. P., and I. M. Robinson Some nutritional characteristics of predominant culturable ruminal bacteria. J. Bacteriol. 84: Bryant, M. P., and N. Small The anaerobic monotrichous butyric acid-producing curved rod-shaped bacteria of the rumen. J. Bacteriol. 72: Bryant, M. P., and N. Small Characteristics of two new genera of anaerobic curved rods isolated from the rumen of cattle. J. Bacteriol. 72: Bryant, M. P., N. Small, C. Bouma, and H. Chu Bacteroides ruminicola n. sp. and Succinimonas amylolytica, the new genus and species. Species of succinic acid-producing anaerobic bacteria of the bovine rumen. J. Bacteriol. 76: Bryant, M. P., N. Small, C. Bouma, and I. M. Robinson Characteristics of ruminal anaerobic cellulolytic cocci and Cillobacterium cellulosolvens n. sp. J. Bacteriol. 76: Bryant, M. P., N. Small, C. Bouma, and I. M. Robinson Studies on the composition of the ruminal flora and fauna of young calves. J. Dairy Sci. 41: Buchanan, R. E., and N. E. Gibbons Bergey's manual of determinative bacteriology, p The Williams & Wilkins Co., Baltimore. 19. Cheng, K.-J., and J. W. Costerton Localization of alkaline phosphatase in three gram-negative rumen bacteria. J. Bacteriol. 116: Cheng, K.-J., and J. W. Costerton Ultrastructure of Butyrivibrio fibrisolvens: a gram-positive bacterium? J. Bacteriol 129: Cheng, K.-J., R. Hironaka, and J. W. Costerton The release of bacterial alkaline phosphatase in the rumen of cattle fed a feedlot bloat-provoking diet and hay diet. Can. J. Microbiol. 22: Cheng, K.-J., R. Hironaka, G. A. Jones, T. Nicas, and J. W. Costerton Frothy feedlot bloat in cattle: production of extracellular polysaccharides and development of viscosity in cultures of Streptococcus bovis. Can. J. Microbiol. 22: Cheng, K.-J., J. M. Ingram, and J. W. Costerton Alkaline phosphatase localization and spheroplast formation of Pseudomonas aeruginosa. Can. J. Microbiol. 16: Cheng, K.-J., J. M. Ingram, and J. W. Costerton Interactions of alkaline phosphatase and the cell wall of Pseudomonas aeruginosa. J. Bacteriol. 107: Cheng, K.-J., G. A. Jones, F. J. Simpson, and M. P. Bryant Isolation and identification of rumen APPL. ENVIRON. MICROBIOL. bacteria capable of anaerobic rutin degradation. Can. J. Microbiol. 15: Costerton, J. W., and K.-J. Cheng The role of the bacterial cell envelope in antibiotic resistance. J. Antimicrob. Chemother. 1: Costerton, J. W., and I. Marks Localization of enzymes in prokaryotic cells, p In M. A. Hayat (ed.), Electron microscopy of enzymes: principles and methods, vol. 5. Van Nostrand Reinhold Co., New York. 28. Eadie, J. M., and S. 0. Mann Development of the rumen microbial population: high starch diets and instability, p In A. T. Phillipson (ed.), Physiology of digestion and metabolism in the ruminant. Oriel Press, Newcastle Upon Tyne, England. 29. Hungate, R. E The anaerobic mesophilic cellulolytic bacteria. Bacteriol. Rev. 14: Hungate, R. E The rumen and its microbes. Academic Press Inc., New York. 31. Ingram, J. M., K.-J. Cheng, and J. W. Costerton Alkaline phosphatase of Pseudomonas aeruginosa: the mechanism of secretion and release of the enzyme fromwhole cells. Can. J. Microbiol. 19: Lindsay, S. S., B. Wheeler, K. E. Sanderson, J. W. Costerton, and K.-J. Cheng The release of alkaline phosphatase and of lipopolysaccharide during the growth of rough and smooth strains of Salmonella typhimurium. Can. J. Microbiol. 19: MacAlister, T. J., J. W. Costerton, L. Thompson, J. Thompson, and J. M. Ingram Distribution of alkaline phosphatase within the periplasmic space of gram-negative bacteria. J. Bacteriol. 111: Porschen, R. K., and E. H. Spaulding Phosphatase activity of anaerobic organisms. Appl. Microbiol. 27: Rangam, C. M., and S. M. Katdare Phosphatase activity of Staphylococci as an indication of their pathogenicity. Indian J. Med. Sci. 8: Rogosa, M Transfer of Peptostreptococcus elsdenii Gutierrez et al. to a new genus, Megasphaera [M. elsdenii [Gutierrez et al.] comb. nov.]. Int. J. Syst. Bacteriol. 21: Scott, H. W., and B. A. Dehority Vitaniin requirements of several cellulolytic rumen bacteria. J. Bacteriol. 89: Thompson, L. M. M., and R. A. MacLeod Biochemical localization of alkaline phosphatase in the cell wall ofa marine pseudomonad. J. Bacteriol. 117: Tsai, C.-G., and G. A. Jones Isolation and identification of rumen bacteria capable of anaerobic phloroglucinol degradation. Can. J. Microbiol. 21: Wolf, P. L., E. Von der Muehll, and M. Ludwick A new test to differentiate Serratia from Enterobacter. Am. J. Clin. Pathol. 56: