Streptococcus Bovis HC5 Research Articles

Stimulation of Bovicin HC5 Production and Selection of Improved Bacteriocin-Producing Streptococcus equinus HC5 Variants

Bovicin HC5 is a peptide that has inhibitory activity against various pathogenic microorganisms and food spoilage bacteria. Aiming to improve the productivity of this bacteriocin, we evaluated several potential factors that could stimulate the synthesis of bovicin HC5 and selected variants of Streptococcus equinus (Streptococcus bovis) HC5 with enhanced bacteriocin production by adaptive laboratory evolution (ALE). The highest production of the bacteriocin (1.5-fold) was observed when Strep. equinus HC5 was cultivated with lactic acid (100mmol/L). For the ALE experiment, Strep. equinus HC5 cells were subjected to acid-shock (pH3.0 for 2h) and maintained in continuous culture for approximately 140 generations (40days) in media with lactic acid (100mmol/L) and pH-controlled at 5.5 ± 0.2. An adapted variant was selected showing a distinct phenotype (sedimentation, pigmentation) compared with the parental strain. Bacteriocin production increased 2-fold in this adapted Strep. equinus HC5 variant, which appears to be associated with changes in the cell envelope of the adapted variant and enhanced bacteriocin release into the culture media. In addition, the adapted variant showed higher levels of expression of all bovicin HC5 biosynthetic genes compared with the parental strain during the early and late stages of growth. Results presented here indicate that ALE is a promising strategy for selecting strains of lactic acid bacteria with increased production of bacteriocins.

LIKE IT ACID AND POOR: A STUDY OF ABIOTIC FACTORS INFLUENCING Streptococcus bovis HC5 GROWTH AND BACTERIOCIN PRODUCTION

This study aimed to investigate the effect of pH, temperature, growth atmosphere and nutrient availability on bovicin HC5 production by Streptococcus bovis HC5. S. bovis HC5 grew well in complex and basal media under aerobic and anaerobic conditions, but greater bacteriocin yields were recovered from anaerobic cultures. Lactate production and glucose consumption increased if S. bovis HC5 cells were cultivated at pH 7.0 and at 45 ºC, but higher bovicin HC5 activity was recovered from cells grown in acidic conditions and at lower temperatures (39 ºC). Cultures maintained under continuous CO2 flow showed faster growth rates in basal media, but bacteriocin production was always higher if S. bovis was cultivated in anaerobic sealed tubes. These results suggest that acidic pH and anoxic conditions favor bovicin HC5 production by S. bovis HC5. S. bovis HC5 is a unique lactic acid bacterium in its ability to grow and produce high amounts of a potentially useful bacteriocin in simple media. Considering the constrains for bacteriocin production at commercial scale, it appears that bovicin HC5 production could be achieved at lower costs compared to other bacteriocins from lactic acid bacteria.

Draft Genome Sequence of Streptococcus equinus (Streptococcus bovis) HC5, a Lantibiotic Producer from the Bovine Rumen

Streptococcus equinus (Streptococcus bovis) HC5 is a bacteriocinogenic lactic acid bacterium with simple growth requirements. The draft genome sequence of S. equinus HC5 consists of 1,846,241 bp, with a G+C content of 37.04%. In silico analysis indicated that S. equinus HC5 might be useful to control bacteria that are detrimental to livestock animals.

English

The antimicrobial effect of bovicin HC5, a bacteriocin produced by Streptococcus bovis HC5, associated with chelating agents or surfactants was investigated against Salmonella Typhimurium. The microorganism grown in brain and heart infusion broth (BHI) was treated with bovicin HC5 at concentrations of 50, 100, 150 and up to 200 AU.mL-1; associated with chelating agents, or surfactants and growth was assessed by absorbance at 630 nm. Bovicin HC5 did not affect the growth of the microorganism, but when combined with chelating agents or surfactants there was inhibition or reduction of growth. The association of bovicin HC5 with EDTA promoted the highest growth inhibition and bactericidal effect after 2.5 h. Gluconic acid, nitrile-tri-acetic acid, diethanolamine, sodium citrate, lactic acid, ramnolipid and Tween 80 reduced the specific growth rate of Salmonella, in at least one of the concentrations of the bovicin HC5 tested. Therefore, the bactericidal effect of bovicin HC5 against Salmonella Typhimurium to is augment in the presence of chelating agents and surfactants, which possibly act in the destabilization of the bacterial outer membrane, which allows the action of the bacteriocin on cell membranes. Key words: Bovicin HC5, bacteriocin, Salmonella Typhimurium.

Chemical composition and nutrient degradability in elephant grass silage inoculated with Streptococcus bovis isolated from the rumen

The objective of the present study was to assess the chemical and bromatological composition and in situ degradability of elephant grass silages inoculated with Streptococcus bovis isolated from cattle rumen. A complete randomized design was used with four treatments and six replications: elephant grass silage, elephant grass silage inoculated with 10(6) CFU/g Streptococcus bovis JB1 strains; elephant grass silage inoculated with 106 CFU/g Streptococcus bovis HC5 strains; elephant grass silage inoculated with 106 CFU/g Enterococcus faecium with six replications each. The pH and ammoniacal nitrogen values were lower (P<0.05) for the silages inoculated with Streptococcus bovis JB1 and HC5, respectively. The silage inoculated with Streptococcus bovis had a higher crude protein content (P<0.05) and there were no differences for the fiber contents in the silage. The (a)soluble fraction degradability, especially in the silages inoculated with Streptococcus bovis JB1 and HC5, had higher values, 30.77 and 29.97%, for dry matter and 31.01 and 36.66% for crude protein, respectively. Inoculation with Streptococcus bovis improved the fermentation profile, protein value and rumen degradability of the nutrients.

Silage fermentation and chemical composition of elephant grass inoculated with rumen strains of Streptococcus bovis

The objective of this study was to evaluate the effects of Streptococcus bovis JB1 and HC5 on the fermentation and chemical composition of elephant grass ensiled in laboratory silos. A complete randomized design was used with four treatments: elephant grass silage, elephant grass silage inoculated with 106CFU/g S. bovis JB1 strains; elephant grass silage inoculated with 106CFU/g S. bovis HC5 strains; elephant grass silage inoculated with 106CFU/g Enterococcus faecium, and six replications. The highest number of enterobacteria, fungi and yeast occurred in the control and elephant grass silages inoculated with E. faecium (P<0.05). The silage inoculated with Streptococcus bovis JB1 and HC5 presented the lowest enterobacteria, fungi and yeast populations and the highest number of lactic acid bacteria, 10.03 and 9.97logCFU/g, respectively. The pH and ammoniac nitrogen values were low (P<0.05) in the treatments with Streptococcus bovis JB1 and HC5, respectively. The observed pH values were 3.83 and 3.96 in the treatments with S. bovis JB1 and HC5, respectively, lower than the observed value of 4.01 in the control treatment. The silages inoculated with S. bovis JB1 and HC5 presented the highest (P<0.05) lactic acid values, 67.8 and 66.2g/kg DM, respectively, and the lowest acetic, butyric and propionic acid values. Inoculation with Streptococcus bovis resulted in a higher (P<0.05) crude protein content in the silages, highest dry matter and crude protein recovery and lowest losses through gases and effluents. Treatments with S. bovis JB1 and HC5 presented values of 93.94 and 92.45 for dry matter recovery. There were no statistical differences (P>0.05) for the other chemical characteristics. Both S. bovis JB1 and HC5 improved the fermentation profile, reduced nutrient losses and increased the nutritional value of the elephant grass silage.

Ruminal degradability of Guinea grass silage inoculated with Streptoccocus bovis isolated from bovine rumen combined or not with com wheat bran

This experiment aimed to evaluate the ruminal degradability in situ of the dry matter (DM), crude protein (CP), neutral detergent fiber (NDF) and acid detergent fiber (ADF) of Guinea grass silages. The experimental design was completely randomized with six treatments and six repetitions: Guinea grass silage, Guinea grass silage inoculated with 10% wheat bran, Guinea grass silage inoculated with 106 cfu/g of strains of Streptococcus bovis JB1; Guinea grass silage inoculated with 106 cfu/g of strains of Streptococcus bovis JB1 plus 10% of wheat bran; Guinea grass silage inoculated with 106 cfu/g of strains of Streptococcus bovis HC5; Guinea grass silage inoculated with 106 cfu/g of strains of Streptococcus bovis HC5 plus 10% of wheat bran. The silages inoculated with Streptococcus bovis and with added wheat bran resulted in higher values of ruminal degradability of DM, 32.76% and 32.17%, and of crude protein 38.28% and 37.89%. For the effective and potential degradability of DM, CP, NDF and ADF the highest values occurred for silages in these silages as well, in all passage rates. There is a similarity between the responses of Streptococcus bovis JB1 and the HC5, and the wheat bran enhanced the effectiveness of the microbial additive.

Effects of the Oral Administration of Viable and Heat-Killed Streptococcus bovis HC5 Cells to Pre-Sensitized BALB/c Mice

Antimicrobial peptides have been suggested as an alternative to classical antibiotics in livestock production and bacteriocin-producing bacteria could be added to animal feeds to deliver bacteriocins in the gastrointestinal (GI) tract of ruminant and monogastric animals. In this study, viable (V) and heat-killed (HK) Streptococcus bovis HC5 cells were orally administered to pre-sensitized mice in order to assess the effects of a bacteriocin-producing bacteria on histological parameters and the immune response of the GI tract of monogastric animals. The administration of V and HK S. bovis HC5 cells during 58 days to BALB/c mice did not affect weight gain, but an increase in gut permeability was detected in animals receiving the HK cells. Viable and heat killed cells caused similar morphological alterations in the GI tract of the animals, but the most prominent effects were detected in the small intestine. The oral administration of S. bovis HC5 also influenced cytokine production in the small intestine, and the immune-mediated activity differed between V and HK cells. The relative expression of IL-12 and INF-γ was significantly higher in the small intestine of mice treated with V cells, while an increase in IL-5, IL-13 and TNF-α expression was only detected in mice treated with HK cells. Considering that even under a condition of severe challenge (pre-sensitization followed by daily exposure to the same bacterial immunogen) the general health of the animals was maintained, it appears that oral administration of S. bovis HC5 cells could be a useful route to deliver bacteriocin in the GI tract of livestock animals.

Ingestão e digestibilidade aparente em ovinos alimentados com silagens de capim-elefante inoculadas com Streptococcus bovis

Foram avaliadas a ingestão e a digestibilidade aparente de silagens de capim, utilizando-se 20 ovinos adultos, sem raça definida, castrados, distribuídos em delineamento inteiramente ao acaso, com quatro tratamentos: silagem de capim-elefante; silagem de capim-elefante inoculada com 10(6)UFC/g de()estirpes de Enterococcus faecium; silagem de capim-elefante inoculada com 10(6)UFC/g de estirpes de Streptococcus bovis JB1; e silagem de capim-elefante inoculada com 10(6)UFC/g de()estirpes de S. bovis HC5, e cinco repetições por tratamento. O período de avaliação foi de 21 dias, sendo 15 para adaptação e seis para as coletas. Observou-se diferença (P&lt;0,05) entre o consumo de matéria seca, sendo a silagem-controle a que apresentou os valores mais baixos - 1,43%PV ou 42,79g/kg PV0,75/dia -, enquanto a silagem inoculada com S. bovis HC5 apresentou o melhor consumo de matéria seca, 2,3% PV ou 56,10g/kg PV0,75/dia. Menores valores de digestibilidade da matéria seca ocorreram na silagem-controle, 42,8% da MS, seguida pela silagem de capim-elefante inoculada com E. faecium e S. bovis JB1. Para a digestibilidade aparente da fibra em detergente neutro, houve efeito dos inoculantes (P&lt;0,05) com valores acima de cinco pontos percentuais em relação à silagem inoculada, 54,7%.

Role of lipid II and membrane thickness in the mechanism of action of the lantibiotic bovicin HC5.

Lantibiotics are antimicrobial peptides produced by Gram-positive bacteria, nisin being the most well-known member. Nisin inhibits peptidoglycan synthesis and forms pores at sensitive membranes upon interaction with lipid II, the essential bacterial cell wall precursor. Bovicin HC5, a bacteriocin produced by Streptococcus bovis HC5, has the putative N-terminal lipid II binding motif, and we investigated the mode of action of bovicin HC5 using both living bacteria and model membranes, with special emphasis on the role of lipid II. Bovicin HC5 showed activity against Staphylococcus cohnii and Staphylococcus warneri, but bovicin HC5 hardly interfered with the membrane potential of S. cohnii. In model membranes, bovicin HC5 was not able to cause carboxyfluorescein release or proton influx from DOPC vesicles containing lipid II. Bovicin HC5 blocked lipid II-dependent pore formation activity of nisin, and a high-affinity interaction with lipid II was observed (apparent binding constant [K(a)] = 3.1 × 10(6) M(-1)), with a 1:1 stoichiometry. In DOPC vesicles containing lipid II, bovicin HC5 was able to assemble with lipid II into a prepore-like structure. Furthermore, we observed pore formation activity of bovicin HC5, which was stimulated by the presence of lipid II, in thin membranes. Moreover, bovicin HC5 induced the segregation of lipid II into domains in giant model membrane vesicles. In conclusion, bovicin HC5 has a primary mode of action similar to that of nisin, but some differences regarding the pore-forming capacity were demonstrated.

The effect of carbon and nitrogen sources on bovicin HC5 production byStreptococcus bovisHC5

To investigate the effect of media composition and agroindustrial residues on bovicin HC5 production by Streptococcus bovis HC5. Batch cultures of S. bovis HC5 were grown in basal medium containing different carbon and nitrogen sources. The activity of cell-free and cell-associated bovicin HC5 was determined in culture supernatants and acidic extracts obtained from cell pellets, respectively. Streptococcus bovis HC5 produced bovicin using a variety of carbon and nitrogen sources. The highest specific activity was obtained in media containing 16 g l(-1) of glucose, after 16 h of incubation. The peak in cell-free and cell-associated bovicin HC5 activity was detected when S. bovis HC5 cultures reached stationary phase. The bovicin HC5 specific activity and bacterial cell mass increased approximately 3-fold when yeast extract and trypticase (0.5 and 1.0 g l(-1), respectively) were added together to the basal medium. Streptococcus bovis HC5 cultures produced bovicin HC5 in cheese whey and sugar cane juice and maximal volumetric productivity was obtained after 12 h of incubation. Streptococcus bovis HC5 is a versatile lactic acid bacterium that can utilize several carbon and nitrogen sources for bovicin HC5 production. This bacterium could be a useful model to study bacteriocin production in the rumen ecosystem. The use of agroindustrial residues as carbon sources could have an economical impact on bovicin HC5 production. To our knowledge, this is the first report to show the use of sugar cane juice for bacteriocin production by lactic acid bacteria.

Bovicin HC5 inhibits wasteful amino acid degradation by mixed ruminal bacteriain vitro

Streptococcus bovis HC5 produces a broad spectrum lantibiotic (bovicin HC5) that inhibits pure cultures of hyper ammonia-producing bacteria (HAB). Experiments were preformed to see if: (1) S. bovis HC5 cells could inhibit the deamination of amino acids by mixed ruminal bacteria taken directly from a cow, (2) semi-purified bovicin was as effective as S. bovis HC5 cells, and 3) semi-purified and the feed additive monensin were affecting the same types of ammonia-producing ruminal bacteria. Because purified and semi-purified bovicin HC5 was as effective as S. bovis HC5 cells, it appeared that bovicin HC5 was penetrating the cell membranes of HAB before it could be degraded by peptidases and proteinases. Mixed ruminal bacteria that were successively transferred and enriched nine times with trypticase did not become significantly more resistant to either bovicin HC5 (50 AU mL(-1)) or monensin (5 microM), and amplified rDNA restriction analysis indicated that bovicin HC5 and monensin appeared to be selecting against the same types of bacteria.

The ability of non-bacteriocin producing Streptococcus bovis strains to bind and transfer bovicin HC5 to other sensitive bacteria

Streptococcus bovis HC5 produces a broad spectrum lantibiotic (bovicin HC5), but S. bovis JB1 does not have antimicrobial activity. Preliminary experiments revealed an anomaly. When S. bovis JB1 cells were washed in stationary phase S. bovis HC5 cell-free culture supernatant, the S. bovis JB1 cells were subsequently able to inhibit hyper-ammonia producing ruminal bacteria ( Clostridium sticklandii, Clostridium aminophilum and Peptostreptococcus anaerobius). Other non-bacteriocin producing S. bovis strains 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 the incubation period was 24 h. Acidic NaCl treatment (100 mM, pH 2.0) liberates half of the bovicin HC5 from S. bovis HC5, but it did not prevent bovicin HC5 from binding to S. bovis JB1. Acidic NaCl liberated some bovicin HC5 from S. bovis JB1, but the decrease in activity was only 2-fold. Bovicin HC5 is a positively 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.

The activity and stability of cell-associated activity of bovicin HC5, a bacteriocin from Streptococcus bovis HC5

Streptococcus bovis HC5 cultures released a broad spectrum lantibiotic, bovicin HC5, into the cell-fee culture supernatant after they reached stationary phase, but most of the antibacterial activity remained cell-associated. Cell-associated bovicin HC5 was more resistant to degradation by Pronase E than the cell-free activity. Acidic NaCl (pH 2.0, 100 mM) did not release all of the cell-associated activity, and cells that were sequentially treated with acidic NaCl and Pronase E still had antibacterial activity. Cell-associated activity retained after acidic NaCl treatment was still able to catalyze potassium efflux from S. bovis JB1, a sensitive strain. These results indicate that cell-associated bovicin HC5 is more active and stable than cell-free bovicin HC5.

The Effect of Calcium and Magnesium on the Activity of Bovicin HC5 and Nisin

Some Gram-positive bacteria produce small peptides (bacteriocins) that have antimicrobial activity, but many bacteria can become bacteriocin resistant. Bovicin HC5, a lantibiotic produced by Streptococcus bovis HC5, has the ability to inhibit nisin-resistant bacteria. Because nisin resistance has in many cases been correlated with an alteration of lipoteichoic acids or the polar head groups of membrane phospholipids, we decided to examine the effect of divalent cations on nisin and bovicin HC5 activity. Both bacteriocins catalyzed potassium efflux from S. bovis JB1, a non-bacteriocin-producing strain. The addition of large amounts (100 mM) of calcium or magnesium increased the ability of S. bovis JB1 to bind Congo red (an anionic dye) and counteracted bacteriocin-mediated potassium loss. Calcium was more effective than magnesium in decreasing nisin activity, but the reverse was observed with bovicin HC5. Nisin-resistant S. bovis JB1 cells bound three times as much Congo red as nisin-sensitive cells, and this result is consistent with the idea that changes in cell surface charge can be a mechanism of bacteriocin resistance. The nisin-resistant cells were less susceptible to bovicin HC5, but bovicin HC5 still caused a 50% depletion of intracellular potassium. These results indicate that nisin and bovicin HC5 react differently with the cell surfaces of Gram-positive bacteria.

Factors affecting the activity of bovicin HC5, a bacteriocin from Streptococcus bovis HC5: release, stability and binding to target bacteria

To determine the factors affecting the release, stability and binding of bovicin HC5 to sensitive bacteria. Stationary phase Streptococcus bovis HC5 cultures had little cell-free bovicin HC5 activity until the final pH was <5.0, and even more bacteriocin was released by treatment with acidic NaCl (pH 2.0, 100 mmol l(-1)). Cultures grown with Tween 80 had more cell-free bovicin HC5 than untreated controls, but this nonionic detergent enhanced activity rather than release. Bovicin HC5 binding to S. bovis JB1 (a susceptible strain) was greater at pH values <6.0. Bovicin HC5 bound other sensitive Gram-positive bacteria, but not Gram-negative species. Cultures retained most of their activity for 35 days, but only if the final pH was <5.6. If the final pH was >5.6, peptidases destroyed much of the activity. Bovicin HC5 remains cell associated until the culture pH is <5.0, but it can be easily dissociated from the cell surface by acidic NaCl. It is highly stable in acidic environments and only binds sensitive bacteria at pH values <6.0. Streptococcus bovis HC5 does not have generally regarded as safe status. However, bovicin HC5 has a broad spectrum of activity and sensitive bacteria do not become resistant. Based on these results, bovicin HC5 may be a useful bacteriocin model.

The effect of pH and a bacteriocin (bovicin HC5) on Clostridium sporogenes MD1, a bacterium that has the ability to degrade amino acids in ensiled plant materials

Fresh plant materials can be fermented and preserved as silage for cattle, but clostridia that deaminate amino acids increase pH. If the pH of the silage rises, spoilage microorganisms proliferate, and undesirable products accumulate. Rod-shaped, anaerobic bacteria with spores were isolated from fresh alfalfa, fresh corn, and silages. Strain MD1 had the highest specific activity of amino acid deamination, and it was most closely related to Clostridium botulinum A and B. However, because strain MD1 did not produce a toxin, it was classified as Clostridium sporogenes. Washed cell suspensions of C. sporogenes MD1 had specific activities as great as 690 nmol ammonia mg protein −1 min −1, and this rate did not decrease until the pH was less than 4.5. Batch cultures of C. sporogenes MD1 did not initiate growth if the initial pH was less than 5.0, but continuous cultures (0.1 h −1 dilution rate) persisted until the pH in the culture vessel was 4.6. When C. sporogenes MD1 was co-cultured with a bacteriocin-producing Streptococcus bovis HC5, ammonia production was greatly reduced. The ability of S. bovis HC5 to inhibit strain MD1 was pH-dependent. When the pH was 5.5 or less, strain MD1 could no longer be detected. These latter results support the idea that bacteriocin-producing bacteria may be used to improve silage quality.

Inhibition of Listeria monocytogenes by bovicin HC5, a bacteriocin produced by Streptococcus bovis HC5

Cattle can be infected with Listeria monocytogenes by consuming contaminated plant materials, soil or silage, and farmers have sought ways of preventing this contamination. Recent work indicated that Streptococcus bovis HC5 produced a bacteriocin (bovicin HC5) that could inhibit a variety of gram-positive bacteria, and we examined the ability of bovicin HC5 to inhibit 10 strains of L. monocytogenes that had been isolated from plant materials, soil, silage and infected cattle. Growth experiments indicated that all of the L. monocytogenes strains were inhibited by 100 activity units (AU) of bovicin HC5 ml −1. L. monocytogenes cultures that were transferred with sublethal doses (12.5 AU ml −1) could be adapted in stepwise fashion to higher doses of bovicin HC5. However, even ‘adapted’ cultures did not grow if 400 AU ml −1 was added. The effect of bovicin HC5 on L. monocytogenes was bactericidal, and viability decreased 5–7 logs after only 2 h of exposure. Bovicin HC5 caused a nearly complete efflux of intracellular potassium in 15 min but only if the pH was less than 6.0. When the pH was greater than 6.0, the cells maintained their potassium pool. L. monocytogenes cells that were acid-adapted (final pH of 4.6) were as sensitive to bovicin HC5 as those that were not acid-adapted (final pH of 6.3). These results support the idea that bovicin HC5 could be effective in controlling listeria in contaminated silages.

Factors affecting the antibacterial activity of the ruminal bacterium, Streptococcus bovis HC5.

Streptococcus bovis HC5 inhibits a variety of S. bovis strains and other Gram-positive bacteria, but factors affecting this activity had not been defined. Batch culture studies indicated that S. bovis HC5 did not inhibit S. bovis JB1 (a non-bacteriocin-producing strain) until glucose was depleted and cells were entering stationary phase, but slow-dilution-rate, continuous cultures (0.2 h(-1)) had as much antibacterial activity as stationary-phase batch cultures. Because the activity of continuous cultures (0.2-1.2 h(-1)) was inversely related to the glucose consumption rate, it appeared that the antibacterial activity was being catabolite repressed by glucose. When the pH of continuous cultures (0.2 h(-1)) was decreased from 6.7 to 5.4, antibacterial activity doubled, but this activity declined at pH values less than 5.0. Continuous cultures (0.2 h(-1)) that had only ammonia as a nitrogen source had antibacterial activity, and large amounts of Trypticase (10 mg ml(-1)) caused only a 2.0-fold decline in the amount of HC5 cell-associated protein that was needed to prevent S. bovis JB1 growth. Because S. bovis HC5 was able to produce antibacterial activity over a wide range of culture conditions, there is an increased likelihood that this activity could have commercial application.

Bovicin HC5, a bacteriocin from Streptococcus bovis HC5.

Previous work indicated that Streptococcus bovis HC5 had significant antibacterial activity, and even nisin-resistant S. bovis JB1 cells could be strongly inhibited. S. bovis HC5 inhibited a variety of Gram-positive bacteria and the spectrum of activity was similar to monensin, a commonly used feed additive. The crude extracts (ammonium sulfate precipitation) were inactivated by Pronase E and trypsin, but the activity was resistant to heat, proteinase K and alpha-chymotrypsin. Most of the antibacterial activity was cell associated, but it could be liberated by acidic NaCl (100 mM, pH 2.0) without significant cell lysis. When glycolysing S. bovis JB1 cells were treated with either crude or acidic NaCl extracts, intracellular potassium declined and this result indicated the antibacterial activity was mediated by a pore-forming peptide. The peptide could be purified by HPLC and matrix-assisted laser desorption ionization time-of-flight analysis indicated that it had a molecular mass of approximately 2440 Da. The terminal amino acid sequence was VGXRYASXPGXSWKYVXF. The unnamed amino acid residues (designated by X) had approximately the same position as dehydroalanines found in some lantibiotics, but samples that were reduced and alkylated prior to Edman degradation did not have cysteine residues. The only other bacteriocin that had significant similarity was the lantibiotic precursor of Streptococcus pyogenes SF370, but the identity was only 55%. Based on these results, the bacteriocin of S. bovis HC5 appears to be novel and the authors now designate it as bovicin HC5.