Anaerobe 15 (2009) 168–172

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Anaerobe

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Physiology and microbial chemistry The ability of non-bacteriocin producing bovis strains to bind and transfer bovicin HC5 to other sensitive bacteriaq

Bruno M. Xavier a, James B. Russell a,b,* a Department of Microbiology, Cornell University, 157A Wing Hall, Ithaca, NY 14853, USA b Agricultural Research Service, USDA, Ithaca, NY 14853, USA article info abstract

Article history: Streptococcus bovis HC5 produces a broad spectrum lantibiotic (bovicin HC5), but S. bovis JB1 does not Received 21 May 2008 have antimicrobial activity. Preliminary experiments revealed an anomaly. When S. bovis JB1 cells were Received in revised form washed in stationary phase S. bovis HC5 cell-free culture supernatant, the S. bovis JB1 cells were 7 August 2008 subsequently able to inhibit hyper-ammonia producing ruminal ( sticklandii, Clos- Accepted 15 October 2008 tridium aminophilum and anaerobius). Other non-bacteriocin producing S. bovis strains Available online 3 January 2009 also had the ability to bind and transfer semi-purified bovicin HC5. Bovicin HC5 that was bound to S. bovis JB1 was much more resistant to Pronase E than cell-free bovicin HC5, but it could be inactivated if Keywords: Streptococcus bovis the incubation period was 24 h. Acidic NaCl treatment (100 mM, pH 2.0) liberates half of the bovicin HC5 Bacteriocins from S. bovis HC5, but it did not prevent bovicin HC5 from binding to S. bovis JB1. Acidic NaCl liberated Bovicin HC5 some bovicin HC5 from S. bovis JB1, but the decrease in activity was only 2-fold. Bovicin HC5 is a posi- tively charged peptide, and the ability of S. bovis JB1 to bind bovicin HC5 could be inhibited by either calcium or magnesium (100 mM). Acidic NaCl-treated S. bovis JB1 cells were unable to accumulate potassium, but they were still able to bind bovicin HC5 and prevent potassium accumulation by untreated S. bovis JB1 cells. Based on these results, bovicin HC5 bound to S. bovis JB1 cells still acts as a pore-forming lantibiotic. Ó 2009 Published by Elsevier Ltd.

1. Introduction known for their ability to become resistant to nisin or other bacteriocins remained sensitive to bovicin HC5, it appeared that Many Gram-positive bacteria produce small peptides (lanti- bovicin HC5 might be a useful lantibiotic [5]. S. bovis JB1 does not biotics) that assemble to form pores in cell membranes [1]. Some produce a lantibiotic, and it has been used as a model organism of lantibiotics have a broad spectrum of antibacterial activity, but bovicin HC5 sensitivity [6–8]. others are species- or even strain-specific. The specificity of lanti- S. bovis HC5 does not liberate significant amounts of cell-free biotics is not entirely clear. Breukink et al. [2] demonstrated that bovicin HC5 until it reaches a stationary phase [9], but competition nisin bound lipid II during its incorporation into the cell membrane. studies indicated that exponentially growing S. bovis HC5 cells However, most Gram-positive bacteria have lipid II and it should be could inhibit S. bovis JB1 [10]. Cell-associated bovicin HC5 can be noted that nisin, the most widely used commercial bacteriocin, is liberated from the cell-surface of S. bovis HC5 by acidic sodium a broad rather than narrow spectrum lantibiotic [1]. chloride (pH 2.0, 100 mM), and divalent cations bind to the cell- Previous work indicated that approximately half of the Strep- surface of S. bovis JB1 and cause resistance to bovicin HC5 [4,6]. tococcus bovis strains isolated from the rumen had antibacterial These latter results indicate that cell-surface charge is an important activity [3], and a strain designated as HC5 produced a broad feature for bovicin HC5 sensitivity and its release from S. bovis HC5. spectrum, positively charged lantibiotic [4]. Because bacteria Clostridium sticklandii SR, Clostridium aminophilum and Peptos- treptococcus anaerobius are hyper-ammonia bacteria (HAB) that are even more sensitive to bovicin HC5 than S. bovis JB1 [4], and they q Mandatory disclaimer: ‘‘Proprietary or brand names are necessary to report have a different pattern of energy source utilization. HAB do not factually on available data; however, the USDA neither guarantees nor warrants the utilize carbohydrates [11] while S. bovis strains use hexoses and do standard of the product, and the use of the name by the USDA implies no approval not ferment amino acids [12]. This difference allowed us to examine of the product, and exclusion of others that may be suitable’’. the ability of S. bovis JB1 to bind bovicin HC5 and transfer it to HAB. * Corresponding author. Department of Microbiology, Cornell University, 157A Wing Hall, Ithaca, NY 14853, USA. Tel.: þ1 607 255 4508; fax: þ1 607 255 3904. Subsequent experiments were designed to describe the nature of E-mail address: [email protected] (J.B. Russell). this transfer.

1075-9964/$ – see front matter Ó 2009 Published by Elsevier Ltd. doi:10.1016/j.anaerobe.2008.10.002 B.M. Xavier, J.B. Russell / Anaerobe 15 (2009) 168–172 169

2. Materials and methods Yang et al. [18] to produce semi-purified bovicin HC5. Semi-purified bovicin HC5 was obtained by lyophilizing the acidic NaCl extract 2.1. Bacteria, media and growth and resuspending it in sterile distilled water (2 ml, 2500 activity units ml1). The semi-purified preparation was assayed for anti- S. bovis strains [13] were routinely grown under O2-free CO2 at bacterial activity by serially diluting the extract in distilled water 39 C in basal medium containing (per liter): 240 mg K2HPO4, (2-fold increments), and placing each dilution (100 ml) in agar wells 240 mg KHPO4, 520 mg Na2SO4, 480 mg NaCl, 100 mg that had been cut into agar plates inoculated with C. sticklandii SR 6 1 MgSO4$7H2O, 64 mg CaCl2$2H2O, 600 mg cysteine hydrochloride, (10 cells ml ) in an anaerobic glove box (Coy Laboratory Products, 1 g Trypticase (BBL Microbiology Systems, Cockeysville, MD, USA), Ann Arbor, MI). Activity units (expressed per milliliter) were vitamins and minerals [14]. The medium was adjusted to pH 6.7 calculated from the reciprocal of the highest serial dilution showing with NaOH and autoclaved for 20 min. After the sterile medium had a visible zone of clearing. 1 cooled to room temperature, sterile Na2CO3 (4 g l ) was added as a buffer. Cultures were grown in 18 150 mm tubes that were 2.6. Potassium accumulation sealed with butyl rubber stoppers. Glucose (4 mg ml1, final concentration) was added to the basal medium after it had been Stationary phase S. bovis JB1 cells were washed and incubated in autoclaved. Growth was monitored via changes in the optical basal medium lacking Trypticase and yeast extract (39 C, 60 min). density (1 cm cuvette, 600 nm, Gilford 260 spectrophotometer, The washed suspensions were then centrifuged (13,000 g, 5 min) Oberlin, OH). C. sticklandii, C. aminophilum and P. anaerobius were through silicone oil as previously described [4]. The cell pellets grown in a similar fashion, except that additional Trypticase were removed with dog nail clippers, digested in 3 N HNO3 (25 C, (20 mg ml1) was substituted for glucose. 24 h), and the insoluble cell material was removed by centrifuga- tion (13,000 g, 1 min). Potassium concentration was determined 2.2. Co-incubation experiments by flame photometry (Cole-Parmer 2655-00 Digital Flame Analyzer, Cole-Parmer Instruments). S. bovis JB1 cells were ener- S. bovis cultures were grown in basal medium until they reached gized by adding glucose (20 mM) and incubating for 30 min prior to the stationary phase (16 h of incubation, 4 mg glucose ml1, final centrifugation through silicone oil. In some cases, untreated S. bovis pH 6.4), and the cells were harvested by centrifugation (4000 g, JB1 cells were co-incubated with S. bovis JB1 cells that had been 5 C, 15 min). Cell-free supernatants were removed from the cell treated with acidic NaCl and subsequently allowed to bind semi- pellets. S. bovis HC5 supernatant was retained and passed through purified bovicin HC5. a 0.45 mm sterile membrane filter (Millipore, Bedford, MA). Non- bacteriocin producing S. bovis cells were resuspended in sterile 3. Results S. bovis HC5 supernatant (15 min, 39 C) and washed with basal medium lacking nitrogen or energy sources. S. bovis cells that had Preliminary experiments indicated that bovicin HC5 was been treated with the S. bovis HC5 supernatant were re-harvested bacteriostatic (but not bactericidal) against S. bovis JB1 and as little by centrifugation and washed again in the basal medium lacking as 20 AU ml1 inhibited growth (data not shown). C. sticklandii SR, nitrogen or energy sources. Bovicin HC5-treated cell pellets were P. anaerobius C and C. aminophilum F (1% v/v inoculum) grew well in resuspended in basal medium containing Trypticase (20 mg ml1), the basal medium that was supplemented with Trypticase and the tubes were inoculated with HAB (1% v/v). After 16 h of (20 mg ml1), and their deamination activities produced more than incubation (39 C), the S. bovis and HAB cells were harvested by 10 mM ammonia after 16 h of incubation (Fig.1). When a tri-culture centrifugation, and the cell-free culture supernatant was analyzed of all HAB was used, the ammonia production was approximately for ammonia. In some cases, the non-bacteriocin producing S. bovis 40 mM. If S. bovis JB1 cells (160 mg protein ml1 or approximately cells were resuspended in basal medium containing semi-purified 109 viable cells ml1) were added to the basal medium that was bovicin (20 AU ml1, described below) rather than sterile filtered S. supplemented with Trypticase a little increase in ammonia was bovis HC5 culture supernatant. detected, and this ammonia could be subtracted to determine the ammonia that was produced by HAB. None of the HAB grew or 2.3. Ammonia production and cell protein produced ammonia in the sterile filtered, S. bovis HC5 supernatant that was supplemented with Trypticase, and this inhibition was Ammonia production was evaluated using the method of consistent with the ability of S. bovis HC5 to liberate cell-free Chaney and Marbach [15]. Six times as much reagent was used bovicin HC5 after it reaches the stationary phase [9]. to eliminate cysteine inference. S. bovis was harvested by centri- HAB were not inhibited by stationary phase S. bovis JB1 cells. fugation (10,000 g, 15 min, 5 C), and the cell pellets were However, if S. bovis JB1 cells were: (1) harvested by centrifugation, digested with dilute NaOH (0.2 N, 100 C, 10 min). Protein content (2) resuspended in stationary phase S. bovis HC5 cell-free super- was assayed using the Lowry method [16] using serum albumin as natant (30 min, 39 C), (3) washed with basal medium and (4) a standard. added to the basal medium that was supplemented with Trypticase (20 mg ml1), all three of the HAB and the HAB tri-culture lost 2.4. Pronase E treatment much of their ability to grow (<0.1 increased in optical density) and produced little ammonia (Fig. 1). With C. sticklandii SR the inhibi- The sterile S. bovis HC5 supernatant or non-bacteriocin tion was approximately 75%. The ability of non-bacteriocin producing S. bovis cells that had been treated with semi-purified producing S. bovis to bind bovicin HC5 and transfer it to HAB was bovicin HC5 were treated with Pronase E (4 U ml1, Sigma Chemical not restricted to the JB1 strain, and it did not seem to matter if the Co., St. Louis, MO) as previously described [17]. S. bovis had been isolated from the rumen (bovine strains) or the of humans (human strains) (Fig. 2). 2.5. Acidic NaCl and semi-purified bovicin HC5 The idea that non-bacteriocin producing S. bovis strains could bind and transfer bovicin HC5 to HAB was strengthened by the Stationary phase S. bovis cells were treated with acidic NaCl observation that a similar effect could be obtained if semi-purified (100 mM NaCl, pH 2.0, 30 min, 39 C) according to the method of bovicin HC5 was added to the basal medium, and the inhibition was 170 B.M. Xavier, J.B. Russell / Anaerobe 15 (2009) 168–172

50 40

40 30

30

20

20 Ammonia Production (mM) Ammonia Production (mM) 10 10

0 0

SR F C SR, F, & C 0 32 64 96 128 160 S. bovis Cells Added (µg protein ml-1) Fig. 1. The ability of S. bovis JB1 (200 mg protein ml1) to bind and transfer bovicin HC5 to C. sticklandii SR, C. aminophilum F and P. anaerobius C and a tri-culture of all three Fig. 3. A comparison of the relative ability of untreated S. bovis JB1 cells (open circles), HAB. The open bar shows the ammonia production of untreated C. sticklandii SR cells. S. bovis JB1 cells that had been allowed to bind bovicin HC5 (filled circles) and S. bovis The grey bar shows the effect of S. bovis JB1 cells that had been allowed to bind bovicin HC5 cells (triangles) to inhibit the ammonia production of C. sticklandii SR. In each case, HC5. In each case, the HAB inoculum was 2% v/v. the S. bovis cell concentration was increased from 0 to 160 mg protein ml1. Semi- purified bovicin HC5 was 20 AU ml1. dependent on the cell density (Fig. 3). If the density of S. bovis JB1 cells was less than 32 mg protein ml1, little inhibition could be inhibitory as S. bovis HC5 cells. Once again, no inhibition by S. bovis demonstrated, but there was a linear decrease in ammonia JB1 cells was observed unless the cells had been allowed to bind production as the S. bovis JB1 cell density was increased to 160 mg bovicin HC5. When the concentration of the semi-purified bovicin protein ml1. S. bovis JB1 cells that had bound bovicin HC5 were as HC5 was varied from 0 to 10 AU ml1, the S. bovis JB1 cells (160 mgml1) had nearly as much capacity to inhibit C. sticklandii 40 SR as cell-free bovicin HC5 (Fig. 4). Recent work indicated that the cell-associated activity of S. bovis HC5 cells was more resistant to Pronase E (a commercial mixture of peptidases and proteinases) than cell-free bovicin HC5 [17], and a similar effect was observed for bovicin HC5 that had been allowed With With to bind to S. bovis JB1 cells. The cell-free supernatant of S. bovis HC5 30 Bovine Human that was treated with Pronase E for 2 h lost most of its ability to S. bovis S. bovis inhibit the ammonia production of C. sticklandii SR, but bovicin HC5 that was bound to S. bovis JB1 cells resisted a similar treatment (data not shown). The resistance of S. bovis JB1 cell-associated bovicin HC5 to degradation by Pronase E was, however, not 20 complete. If the incubation time with Pronase E was increased to 24 h, even the S. bovis JB1 cell-associated bovicin HC5 lost most of its activity (data not shown). Acidic NaCl (100 mM, pH 2.0) is used to remove bacteriocins

Ammonia Production (mM) from [18]. However, S. bovis JB1 cells that were 10 treated with acidic NaCl still bound bovicin HC5 and transferred it to C. sticklandii SR (Fig. 5). S. bovis JB1 cells that were first treated with bovicin HC5 and then subjected to acidic NaCl were only 2- fold less potent than cells that had not been treated with acidic NaCl. However, if large amounts of divalent cations (100 mM 0

6 magnesium or calcium) were added, the S. bovis JB1 cells FM

1499 9410 completely lost their ability to bind bovicin HC5 and transfer it to 33317 15531 43143 V1387 HAB (data not shown). K27FFA SR alone 581AXY Stationary phase S. bovis JB1 cells that were washed and 1 Fig. 2. The ability of bovine and human S. bovis strains (200 mg protein ml ) to bind incubated in the basal medium lacking nitrogen sources or and transfer bovicin HC5 to C. sticklandii SR. The open bar shows the ammonia glucose had an intracellular potassium concentration of only production of untreated C. sticklandii SR cells. The grey bars show the effect of S. bovis 1 1 cells that had been washed with stationary phase, S. bovis HC5 supernatant. Strain 700 nmol mg protein min , and those treated with acidic NaCl numbers of S. bovis are listed at the bottom. had even less potassium (Fig. 6). When the treated and untreated B.M. Xavier, J.B. Russell / Anaerobe 15 (2009) 168–172 171

40 4000

Without With

) Glucose Glucose -1

30 3000

20 2000 Ammonia Production (mM) 10 1000 Intracellular Potassium (nmol mg protein

0 0 ABC DEF 0246810 Bovicin HC5 Added (AU ml-1) Fig. 6. The intracellular potassium concentration of non-energized, non-treated S. bovis JB1 cells (A), non-energized, acidic NaCl-treated S. bovis JB1 cells that had also Fig. 4. The ability of S. bovis JB1 cells (160 mg protein ml1) to bind semi-purified been allowed to bind semi-purified bovicin HC5 (B) and a combination of cells shown bovicin HC5 and inhibit the ammonia production of C. sticklandii SR (filled circles). The in A and B (C). Parts (D–F) show the same cells, respectively, that were energized with open circles show the same amount of semi-purified bovicin HC5. glucose (20 mM). Open bars show incubations (30 min, 39 C) that were conducted at pH 6.7. Filled bars show incubations (30 min, 39 C) that were conducted at pH 5.5. cells were mixed together (160 mg protein1 ml1, each) the value accumulate potassium at either pH, but they were able to prevent was approximately 350 nmol mg protein1 min1. When glucose the potassium accumulation of untreated cells at pH 5.5. The (20 mM) was added, the untreated cells accumulated potassium mixture of treated and untreated cells was still able to accumulate and had approximately 2400 nmol mg protein1 min1 at either potassium at pH 6.7. pH 6.7 or 5.5. The bovicin and acidic NaCl-treated cells did not 4. Discussion 40 Rumen is a very complex ecosystem that is inhabited by a variety of bacteria, protozoa and fungi, and molecular techniques indicate that the is continuously changing [19]. A variety of Gram-positive ruminal bacteria produce bacteriocins [20], and microbiologists have typically focused on cell-free bacteriocin 30 activity because it can diffuse through agar and create zones of clearing that are observed visually [1]. The role of bacteriocins in ruminal ecology has been stymied by the observation that it is difficult if not impossible to detect cell-free activity using standard methods of agar diffusion [unpublished results]. However, previous 20 work showed that mixed rumen bacteria inhibited from cattle fed a grain-based ration that were harvested by centrifugation inhibi- ted a tri-culture of HAB and decreased the initial rate of ammonia production by 50% [21]. Because bacteria taken from a cow fed

Ammonia Production (mM) timothy hay were unable to inhibit the HAB tri-culture, it appeared 10 that cows fed hay have fewer bacteriocin producing bacteria. This idea was consistent with the observation that cows fed hay have a 2-fold greater specific rate of ammonia production than cows fed mostly grain [21,22]. Many bacteriocin producing, lactic acid bacteria have cell- 0 associated activity that can be released by treatment with acidic 0246810NaCl [18], and this method was used to prepare semi-purified Fold Dilution bovicin HC5 [4]. Competition experiments between S. bovis HC5 and JB1 indicated that bovicin HC5 production allowed HC5 to Fig. 5. The effect of S. bovis JB1 cells (160 mg protein ml1) on the ammonia production dominate the co-cultures even though HC5 had a slower growth of C. sticklandii SR cells. Open triangles show S. bovis JB1 cells that were allowed to bind rate than JB1 and cell-free bovicin HC5 could not yet be detected semi-purified bovicin HC5. Open circles show S. bovis JB1 cells that were treated with [10]. These results support the idea that cell-free bovicin HC5 is acidic NaCl and then allowed to bind semi-purified bovicin HC5. Filled triangles show S. bovis JB1 cells that were allowed to bind semi-purified bovicin HC5 and then were only detected in stationary phase cultures [9], and the cell-associ- treated with acidic NaCl. ated activity seems to be more important [17]. The cell-associated 172 B.M. Xavier, J.B. Russell / Anaerobe 15 (2009) 168–172 bovicin HC5 activity was more resistant to Pronase E, and this bacteria are in most cases resistant to monensin, the most characteristic could be important in the rumen because commonly used feed additive in American rations. HAB have much many ruminal microorganisms produce proteinases [20] and greater deamination activities, can produce as much as 90% of the peptidases [23]. ruminal ammonia [21] and are sensitive to both monensin [11] and The observation that non-bacteriocin producing S. bovis strains bovicin HC5 [4]. Because monensin has been banned by the Euro- could bind bovicin HC5, transfer it to HAB and decrease the growth pean Union, bovicin HC5 may offer an alternative way of decreasing and ammonia production of HAB, is yet another reason why bacte- excess ruminal ammonia [26]. Very recently published work like- riocin activity may be difficult to detect in cell-free ruminal fluid (see wise indicates that the cell-associated bacteriocin of S. bovis HC5 is above). Comparisons of semi-purified bovicin HC5 alone and with more potent and less likely to be degraded by peptidases than the the amount of bovicin HC5 that S. bovis JB1 was able to bind indi- cell-free form [17]. cated that S. bovis JB1 bound large amounts of bovicin HC5, and bovicin HC5-treated S. bovis JB1 cells were approximately as potent as S. bovis HC5 cells. Bovicin HC5 that was bound to S. bovis JB1 was References more resistant to Pronase E than cell-free bovicin HC5, and this observation supports the idea that this type of binding also confers [1] Jack RW, Tagg JR, Ray B. Bacteriocins of gram-positive bacteria. Microbiol Rev 1995;59(2):171–200. increased stability. Further work will be needed to determine the [2] Wiedemann I, Breukink E, van Kraaij C, Kuipers OP, Bierbaum G, de Kruijff B, mechanism of bovicin HC5 transfer from S. bovis JB1 to HAB, but it et al. Specific binding of nisin to the peptidoglycan precursor lipid II combines should be noted that agglutination per se was not readily observed. pore formation and inhibition of cell wall biosynthesis for potent antibiotic activity. J Biol Chem 2001;276(3):1772–9. As previously noted, acidic NaCl extracts large amounts of [3] Mantovani HC, Kam DK, Ha JK, Russell JB. The antibacterial activity and bacteriocins from lactic bacteria [18], but previous work indicated sensitivity of Streptococcus bovis strains isolated from the rumen of cattle. that it was only able to liberate approximately half of the bovicin FEMS Microbiol Ecol 2001;37(3):223–9. [4] Mantovani HC, Hu H, Worobo RW, Russell JB. Bovicin HC5, a bacteriocin from HC5 from S. bovis HC5 cells [17]. Our observation that acidic NaCl- Streptococcus bovis HC5. Microbiology 2002;148(Pt 11):3347–52. treated S. bovis JB1 cells retained large amounts of bovicin HC5 [5] Russell JB, Mantovani HC. The bacteriocins of ruminal bacteria and their indicates that S. bovis might have two different methods of binding. potential as an alternative to antibiotics. J Mol Microbiol Biotechnol Acidic NaCl-resistant binding might be common to all S. bovis. 2002;4(4):347–55. [6] Houlihan AJ, Russell JB. Factors affecting the activity of bovicin HC5, a bacte- Whereas, the acidic NaCl-susceptible binding might be the one riocin from Streptococcus bovis HC5: release, stability and binding to target more closely related to the production and secretion of bovicin HC5 bacteria. J Appl Microbiol 2006;100(1):168–74. by S. bovis HC5. [7] Houlihan AJ, Mantovani HC, Russell JB. Effect of pH on the activity of bovicin HC5, a bacteriocin from Streptococcus bovis HC5. FEMS Microbiol Lett A recent review by Hasper et al. [24] noted that the lantibiotic, 2004;231(1):27–32. nisin, has an ‘‘alternative bactericidal mechanism of action.’’ This [8] Houlihan AJ, Russell JB. The effect of calcium and magnesium on the activity of alternative activity is mediated at the level of lipid II and cell wall bovicin HC5 and nisin. Curr Microbiol 2006;53(5):365–9. [9] Mantovani HC, Russell JB. Factors affecting the antibacterial activity of the synthesis rather than pore formation and the loss of intracellular ruminal bacterium, Streptococcus bovis HC5. Curr Microbiol 2003;46(1):18–23. solutes. Our previous work indicated that acidic NaCl-treated [10] Xavier BM, Russell JB. Bacterial competition between a bacteriocin-producing S. bovis HC5 cells were unable to accumulate potassium, and the and a bacteriocin-negative strain of Streptococcus bovis in batch and contin- uous culture. FEMS Microbiol Ecol 2006;58(3):317–22. activity that remained on the cells after acidic NaCl treatment [11] Paster BJ, Russell JB, Yang CMJ, Chow JM, Woese CR, Tanner R. Phylogeny of the prevented potassium accumulation by S. bovis JB1 [17]. These ammonia-producing ruminal bacteria Peptostreptococcus anaerobius, Clos- results indicated that the cell-associated activity was still acting as tridium sticklandii, and Clostridium aminophilum sp-nov. Int J Syst Bacteriol 1993;43(1):107–10. a pore-forming lantibiotic and not just as an inhibitor of cell wall [12] Russell JB, Robinson PH. Compositions and characteristics of strains of Strep- synthesis. Our current work indicated that acidic NaCl-treated tococcus bovis. J Dairy Sci 1984;67(7):1525–31. S. bovis JB1 cells had approximately the same ability to bind bovicin [13] Kurtovic A, Jarvis GN, Mantovani HC, Russell JB. Ability of lysozyme and 2- HC5 as untreated S. bovis JB1 cells. The acidic NaCl-treated S. bovis deoxyglucose to differentiate human and bovine Streptococcus bovis strains. J Clin Microbiol 2003;41(8):3951–4. JB1 cells were also able to transfer bovicin HC5 to untreated S. bovis [14] Cotta MA, Russell JB. Effect of peptides and amino acids on efficiency of rumen JB1 cells and prevent potassium accumulation at pH 5.5 but not 6.7. bacterial protein-synthesis in continuous culture. J Dairy Sci 1982;65(2):226–34. This latter result is consistent with the observation that bovicin [15] Chaney AL, Marbach EP. Modified reagents for determination of urea and ammonia. Clin Chem 1962;8:130–2. HC5 is much more active at an acidic pH [7]. [16] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Further work will be needed to define more precisely the Folin phenol reagent. J Biol Chem 1951;193(1):265–75. binding of bovicin HC5 to S. bovis JB1 and other non-bacteriocin [17] Xavier BM, Houlihan AJ, Russell JB. The activity and stability of cell-associated activity of bovicin HC5, a bacteriocin from Streptococcus bovis HC5. FEMS producing strains. However, it should be noted that lantibiotics first Microbiol Lett 2008;283(2):162–6. pass through the extracellular glycocalyx that typically carries a net [18] Yang R, Johnson MC, Ray B. Novel method to extract large amounts of negative charge. Most lantibiotics [1], including bovicin HC5 [4] are bacteriocins from lactic acid bacteria. Appl Environ Microbiol 1992;58(10):3355–9. positively charged peptides, and the glycocalyx has been implicated [19] Russell JB. Rumen microbiology and its role in ruminal fermentation. Ithaca, in at least some forms of bacteriocin resistance [25]. Previous work NY: JB Russell Publishing Co.; 2002. indicated that S. bovis JB1 cells that were incubated and washed [20] Brock FM, Forsberg CW, Buchanansmith JG. Proteolytic activity of rumen with large amounts of calcium or magnesium became bovicin HC5- microorganisms and effects of proteinase inhibitors. Appl Environ Microbiol 1982;44(3):561–9. resistant, and this resistance was correlated with an increased [21] Rychlik JL, Russell JB. Mathematical estimations of hyper-ammonia producing ability of the cells to bind Congo red, an anionic dye [8]. Because our ruminal bacteria and evidence for bacterial antagonism that decreases current work indicated that high concentrations of calcium or ruminal ammonia production. FEMS Microbiol Ecol 2000;32(2):121–8. [22] Lana RP, Russell JB, Van Amburgh ME. The role of pH in regulating ruminal magnesium blocked the ability of S. bovis JB1 cells to bind bovicin methane and ammonia production. J Anim Sci 1998;76(8):2190–6. HC5 and transfer it to C. sticklandii SR, it appears that the binding is [23] Wallace RJ, Mckain NA. A survey of peptidase activity in rumen bacteria. J Gen electrostatic. Microbiol 1991;137:2259–64. [24] Hasper HE, Kramer NE, Smith JL, Hillman JD, Zachariah C, Kuipers OP, et al. An The observation that bovicin HC5 either directly or indirectly via alternative bactericidal mechanism of action for lantibiotic peptides that non-bacteriocin producing bacteria such as S. bovis JB1 can inhibit target lipid II. Science 2006;313:1636–7. HAB has practical significance. For many years it was assumed that [25] Mantovani HC, Russell JB. Nisin resistance of Streptococcus bovis. Appl Environ Microbiol 2001;67:808–13. carbohydrate fermenting ruminal bacteria were responsible for the [26] Russell JB, Houlihan A. Ionophore resistance of ruminal bacteria and its wasteful degradation of amino acids in the rumen, but these potential impact on human health. FEMS Microbiol Rev 2002;27:65–74.