Interactions between anaerobic cellulolytic bacteria and fungi in the presence of Methanobrevibacter smithii

A mixed inoculum of cellulolytic rumen bacteria depressed straw degradation by a mixed culture of cellulolytic fungi grown in the presence of Methanobrevibacter smithii. The inhibitory effect appeared to be caused by Ruminococcus albus strain JI and R. flavefaciens strain 007. Ruminococcus albus strain J1 also depressed straw degradation by the fungi, but R. albus strain SY3 and three strains of Bacteroides (Fibrobacter) succinogenes tested showed little or no inhibitory activity. It seems that some ruminococci show competitive or antagonistic activity towards certain rumen fungi.     

Albusin B, a bacteriocin from the ruminal bacterium Ruminococcus albus 7 that inhibits growth of Ruminococcus flavefaciens

An approximately 32-kDa protein (albusin B) that inhibited growth of Ruminococcus flavefaciens FD-1 was isolated from culture supernatants of Ruminococcus albus 7. Traditional cloning and gene-walking PCR techniques revealed an open reading frame (albB) encoding a protein with a predicted molecular mass of 32,168 Da. A BLAST search revealed two homologs of AlbB from the unfinished genome of R. albus 8 and moderate similarity to LlpA, a recently described 30-kDa bacteriocin from Pseudomonas sp. strain BW11M1.     

Thuricin 7: a novel bacteriocin produced by Bacillus thuringiensis BMG1.7, a new strain isolated from soil

AIMS: Detection and identification of new antagonistic activities towards Bacillus cereus and relatives. METHODS AND RESULTS: Twenty Bacillus thuringiensis strains were screened for their capacity to express bacteriocin-like agents. Strain BMG1.7, isolated from soil, showed an antagonistic activity called thuricin 7. Thuricin 7 was active against several species of the genus Bacillus, including three of the four known B. thuringiensis/B. cereus bacteriocin producers, as well as against Streptococcus pyogenes and Listeria monocytogenes strains. Antimicrobial activity was lost after treatment with proteinase K. The active protein had an apparent molecular weight of 11.6 kDa, and was secreted at the end of the exponential growth phase. Thuricin 7 retained 55% of the activity after incubation at 98 degrees C for 30 min. The mode of action of thuricin 7 was shown to be bactericidal and bacteriolytic. CONCLUSION: Thuricin 7 is a novel bacteriocin produced by a newly isolated Bacillus thuringiensis strain BMG1.7. SIGNIFICANCE AND IMPACT OF THE STUDY: The characteristics of thuricin 7 indicate that it is a new bacteriocin which may have interesting biotechnological applications due to its relatively large activity spectrum.     

Characterization and partial purification of a broad spectrum antibiotic AS-48 produced by Streptococcus faecalis

Streptococcus faecalis S-48 produces a broad spectrum antibiotic, active against Gram-positive and Gram-negative bacteria. This substance is produced in solid and liquid media and also in a defined basal medium. It is sensitive to protease, pronase, or trypsin, heating at 70 degrees C, and alkaline pH, but resistant to treatment with lipase, lysozyme, alkaline phosphatase, DNAase, RNAase, acidic or neutral pHs, and also lower temperatures (60 degrees C). Several organic solvents cause precipitation, but not inactivation. This antibiotic has been partially purified by gel filtration and further ion-exchange chromatography. Its molecular weight has been estimated close to 2000. The biological activity of this antagonistic substance against the selected indicator strains, Streptococcus faecalis S-47 and Escherichia coli U-9, is bactericidal. The characterization of this substance, initially classified as a bacteriocin, indicates that it is an antibiotic of peptidic nature. The significance of antibiotic occurrence in group D of the genus Streptococcus is also discussed.     

Bacteriocin-like substances produced by Rhizobium japonicum and other slow-growing rhizobia

Bacteriocin-like substances were commonly produced by slow-growing Rhizobium japonicum and cowpea rhizobia on an L-arabinose medium. Antagonism between strains of R. japonicum was not detected in vitro; however, such strains were often sensitive to some bacteriocins produced by cowpea rhizobia. Inhibitory zones (2 to 8 mm from colony margins), produced by 58 of 66 R. japonicum test strains, were reproducibly detected with Corynebacterium nebraskense as an indicator. Quantitative production was not related to symbiotic properties of effective strains, since nine noninfective strains and one ineffective strain produced bacteriocin. Eight R. japonicum strains that did not produce bacteriocin nevertheless formed effective nodules on soybeans. R. japonicum strains that produced bacteriocin in vitro had no antagonistic effect on nonproducer strains during soybean nodulation. Under controlled conditions, a nonproducer (3I1b135) predominated over a bacteriocin producer (3I1b6) when inoculated at 1:1 and 1:9 ratios. Depending on the particular ratio, up to 38% of the total nodules formed were infected with mixed combinations. The bacteriocin(s) had a restricted host range and antibiotic-like properties which included the ability to be dialyzed and resistance to heat (75 to 80 degrees C, 30 min), Pronase, proteinase K, trypsin, ribonuclease, and deoxyribonuclease. R. japonicum strains representing genetic, serological, cultural, and geographic diversity were differentiated into three groups on the basis of bacteriocin production.     

The use of 16S rRNA-targeted oligonucleotide probes to study competition between ruminal fibrolytic bacteria: development of probes for Ruminococcus species and evidence for bacteriocin production.

A total of six oligonucleotide probes, complementary to the 16S rRNA, were evaluated for quantitative and determinative studies of Ruminococcus albus and Ruminococcus flavefaciens. On the basis of specificity studies, probes for R. albus (probe RAL196) and R. flavefaciens (probe RFL196) were selected to quantitate these species in mixed culture. In combination with a Fibrobacter succinogenes S85 subspecies probe (SUB1) and a domain Bacteria (formerly kingdom Eubacteria) probe (EUB338), they were used to quantitate these species competing in mixed cultures for cellobiose as the carbon source. In dicultures containing R. albus 8 and F. succinogenes S85, competition was not observed. However, R. flavefaciens FD-1 eventually outcompeted F. succinogenes S85 when cellobiose was the substrate. When R. albus 8 and R. flavefaciens FD-1 were grown together on cellobiose medium, R. albus 8 outcompeted R. flavefaciens FD-1, resulting in undetectable R. flavefaciens 16S rRNA only 1 to 3 h after inoculation, suggesting production of an antagonistic compound by R. albus 8 during rapid growth on soluble substrates. Further, when R. albus 8, R. flavefaciens FD-1, and F. succinogenes S85 were grown together in a triculture, R. flavefaciens FD-1 16S rRNA was detectable for only 2 h after inoculation, while R. albus 8 and F. succinogenes S85 showed a similar competition pattern to that of the dicultures. The results show that the Ruminococcus probes were effective in the measurement of relative populations of selected R. albus and R. flavefaciens strains during in vitro competition studies with F. succinogenes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intergeneric Protoplast Fusion between Ruminococcus albus and an Anaerobic Recombinant, FE7

Intergeneric protoplast fusion between Ruminococcus albus, a cellulolytic, gram-positive, anaerobic bacterium (Pc Sm Km), and an anaerobic recombinant, FE7 (Pc Sm Km), having lignin-related compound-degrading activities, was performed under strictly anaerobic conditions to introduce cellulase genes into strain FE7. The fusion frequency varied with different selected markers from 3.0 x 10 to 3.3 x 10. Two fusants, obtained from a synthetic medium with selective pressures of penicillin and streptomycin and with cellooli-gomer as the sole carbon source, were gram-negative rods. One of them, named FE7R2, showed 45 to 47% of the beta-glucosidase and cellobiosidase activities of its parent R. albus and still maintained a level of degradation activity against dehydrodivanillin, a lignin-related compound, of up to 87% of that of the parent strain FE7. To verify that the cellulolytic activities expressed in the fusant FE7R2 originated from R. albus cellulase genes, the beta-glucosidase gene of R. albus was cloned into Escherichia coli HB101 with plasmid pBR322. Cells bearing a recombinant plasmid, pRAII, produced high enzyme activities against both p-nitrophenyl-beta-d-glucoside and p-nitrophenyl-beta-d-cellobioside and could degrade cellobiose to glucose. Southern blot results showed that the cloned DNA fragment could hybridize with chromosomal DNAs of both R. albus and FE7R2, but did not with the chromosomal DNA of FE7, indicating that the beta-glucosidase gene fragment was introduced into the chromosome of FE7R2 from R. albus via the protoplast fusion. The fusant FE7R2 could utilize simultaneously both cellobiose and dehydrodivanillin. These results gave evidence that the fusion product FE7R2 is a recombinant strain between its parents R. albus and FE7. This recombinant has stably kept the above properties for about 2 years.     

Cloning, sequencing, and expression in Escherichia coli of lcnB, a third bacteriocin determinant from the lactococcal bacteriocin plasmid p9B4-6.

On the bacteriocin plasmid p9B4-6 of Lactococcus lactis subsp. cremoris 9B4, a third bacteriocin determinant was identified. The genes encoding bacteriocin production and immunity resided on a 1.2-kb CelII-ScaI fragment and were located adjacent to one of two previously identified bacteriocin operons (M. J. van Belkum, B. J. Hayema, R. E. Jeeninga, J. Kok, and G. Venema, Appl. Environ. Microbiol. 57:492-498, 1991). The fragment was sequenced and analyzed by deletion and mutation analyses. The bacteriocin determinant consisted of two genes which were transcribed as an operon. The first gene (lcnB), containing 68 codons, was involved in bacteriocin activity. The second gene (lciB) contained 91 codons and was responsible for immunity. The specificity of this novel bacteriocin, designated lactococcin B, was different from that of the other two bacteriocins specified by p9B4-6. Part of the nucleotide sequence of the lactococcin B operon was similar to a nucleotide sequence also found in the two other bacteriocin operons of p9B4-6. This conserved region encompassed a nucleotide sequence upstream of the bacteriocin gene and the 5' part of the gene. When the lactococcin B operon was expressed in Escherichia coli by using a T7 RNA polymerase-specific promoter, antagonistic activity could be detected.     

Cloning, sequencing, and expression in Escherichia coli of lcnB, a third bacteriocin determinant from the lactococcal bacteriocin plasmid p9B4-6.

On the bacteriocin plasmid p9B4-6 of Lactococcus lactis subsp. cremoris 9B4, a third bacteriocin determinant was identified. The genes encoding bacteriocin production and immunity resided on a 1.2-kb CelII-ScaI fragment and were located adjacent to one of two previously identified bacteriocin operons (M. J. van Belkum, B. J. Hayema, R. E. Jeeninga, J. Kok, and G. Venema, Appl. Environ. Microbiol. 57:492-498, 1991). The fragment was sequenced and analyzed by deletion and mutation analyses. The bacteriocin determinant consisted of two genes which were transcribed as an operon. The first gene (lcnB), containing 68 codons, was involved in bacteriocin activity. The second gene (lciB) contained 91 codons and was responsible for immunity. The specificity of this novel bacteriocin, designated lactococcin B, was different from that of the other two bacteriocins specified by p9B4-6. Part of the nucleotide sequence of the lactococcin B operon was similar to a nucleotide sequence also found in the two other bacteriocin operons of p9B4-6. This conserved region encompassed a nucleotide sequence upstream of the bacteriocin gene and the 5' part of the gene. When the lactococcin B operon was expressed in Escherichia coli by using a T7 RNA polymerase-specific promoter, antagonistic activity could be detected.     

Identification of the agent from Lactobacillus plantarum KFRI464 that enhances bacteriocin production by Leuconostoc citreum GJ7

AIM: To provide evidence that the production of bacteriocin by lactic acid bacteria can be enhanced by the presence of a bacteriocin-sensitive strain and identify the agent that is responsible for enhancing bacteriocin production. METHODS AND RESULTS: One bacteriocin-producing lactic acid bacterium was isolated from kimchi. The strain GJ7 was designated as Leuconostoc citreum GJ7 based on Gram staining, biochemical properties, and 16S rRNA gene sequencing. The isolate produced a heat- and pH-stable bacteriocin (kimchicin GJ7), which has antagonistic activity against a broad spectrum of micro-organisms. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified kimchicin GJ7 showed a single band of molecular weight c. 3500 Da. Cultures of Leuc. citreum GJ7 in the presence of thermally inactivated kimchicin GJ7-sensitive strains, Lactobacillus plantarum KFRI 464, Lactobacillus delbrueckii KFRI 347, or Leuconostoc mesenteroides KCTC 1628, increased bacteriocin production. This inducing factor was characterized and purified from Lact. plantarum KFRI 464, which showed the greatest enhancement of kimchicin GJ7 activity. The inducing factor was purified using a DEAE (diethyl aminoethyl)-Sephacel column and high-performance liquid chromatography, and yielded a single band of c. 6500 Da. N-terminal sequencing of the inducing factor identified 16 amino acids. The N-terminal sequence of the inducing factor was synthesized and examined for the induction of kimchicin GJ7 activity, and was found to induce activity, but at a level about 10% lower than that of the entire molecule. CONCLUSIONS: The presence of a bacteriocin-sensitive strain, Lact. plantarum KFRI 464, acts as an environmental stimulus to activate the production of kimchicin GJ7 by Leuc. citreum GJ7. The inducing factor from Lact. plantarum KFRI 464 is highly homologous to the 30S ribosomal protein S16 from various micro-organisms. The N-terminal sequence of the inducing factor examined in this study is a very important sequence related to the inducing activity. Nevertheless, the inducing factor may not be part of the ribosomal protein S16 itself. SIGNIFICANCE AND IMPACT OF THE STUDY: We believe that the present study is the first to identify an agent that is produced by one micro-organism and influences bacteriocin production in another. The bacteriocin-enhancing system described in this study could be effectively used to control the growth of other micro-organisms (sensitive cells) in food systems. Moreover, this enhancement of bacteriocin production can be applied usefully in industrial production of natural food preservatives.     

Bacteriocin-Like Substances Produced by Rhizobium japonicum and Other Slow-Growing Rhizobia

Bacteriocin-like substances were commonly produced by slow-growing Rhizobium japonicum and cowpea rhizobia on an L-arabinose medium. Antagonism between strains of R. japonicum was not detected in vitro; however, such strains were often sensitive to some bacteriocins produced by cowpea rhizobia. Inhibitory zones (2 to 8 mm from colony margins), produced by 58 of 66 R. japonicum test strains, were reproducibly detected with Corynebacterium nebraskense as an indicator. Quantitative production was not related to symbiotic properties of effective strains, since nine noninfective strains and one ineffective strain produced bacteriocin. Eight R. japonicum strains that did not produce bacteriocin nevertheless formed effective nodules on soybeans. R. japonicum strains that produced bacteriocin in vitro had no antagonistic effect on nonproducer strains during soybean nodulation. Under controlled conditions, a nonproducer (3I1b135) predominated over a bacteriocin producer (3I1b6) when inoculated at 1:1 and 1:9 ratios. Depending on the particular ratio, up to 38% of the total nodules formed were infected with mixed combinations. The bacteriocin(s) had a restricted host range and antibiotic-like properties which included the ability to be dialyzed and resistance to heat (75 to 80°C, 30 min), Pronase, proteinase K, trypsin, ribonuclease, and deoxyribonuclease. R. japonicum strains representing genetic, serological, cultural, and geographic diversity were differentiated into three groups on the basis of bacteriocin production.     

Luminescent method for the detection of antibacterial activities

A new rapid and sensitive method for the detection of antibacterial activities was based on luminescent indicator strains. Listeria innocua 8811 and Enterococcus faecalis 32 were transformed with plasmid carrying bacterial luciferase genes. Subsequent strains became capable to emit light during the exponential growth phase. The addition of bacteriocin containing culture supernatants to such cultures induced a drop of their light emission which was correlated to the combined antibacterial activity of acid stress and bacteriocin. The detection of antagonistic activity is independent of its mode of action, i.e. bactericidal or bacteriostatic. This method allowed to directly visualize the antagonistic activity of bacteriocin producer strains toward target strains in coculture experiments. However, a control co-culture with non-producing bacteriocin mutant was necessary in order to distinguish between nutrients competition and bacteriocin activity. Finally, five class IIa bacteriocins were purified from culture supernatants of eight strains detected in 3 days from a 120 lactic acid bacteria collection.     

Subcellular distribution of glycanases and related components in Ruminococcus albus SY3 and their role in cell adhesion to cellulose

AIMS: To compare the subcellular distribution of glycanase-related components between wild-type Ruminococcus albus SY3 and an adhesion-defective mutant, to identify their possible contribution to the adhesion process, and to determine their association with cellulosome-like complexes. METHODS AND RESULTS: Cell fractionation revealed that most of the cellulases and xylanases were associated with capsular and cell-wall fractions. SDS-PAGE and gel filtration indicated that most of the bacterial enzyme activity was not integrated into cellulosome-like complexes. The adhesion-defective mutant produced significantly less (5- to 10-fold) overall glycanase activity, and the 'true cellulase activity' appeared to be entirely confined to the cell membrane fractions. Antibodies specific for the cellulosomal scaffoldin of Clostridium thermocellum recognized a single 240 kDa band in R. albus SY3. CONCLUSIONS: The adhesion-defective mutant appeared to be blocked in exocellular transport of enzymes involved in true cellulase activity. A potential cellulosomal scaffoldin candidate was identified in R. albus SY3. SIGNIFICANCE AND IMPACT OF THE STUDY: Several glycanase-related proteins and more than one mechanism appear to be involved in the adhesion of R. albus SY3 to cellulose.     

Partial Purification and Characterization of a Bacteriocin DT24 Produced by Probiotic Vaginal Lactobacillus brevis DT24 and Determination of its Anti-Uropathogenic Escherichia coli Potential

The emergence of antibiotic resistance has increased the interest for finding new antimicrobials in the past decade. Probiotic Lactic acid bacteria producing antimicrobial proteins like bacteriocin can be excellent agents for development as novel therapeutic agents and complement to conventional antibiotic therapy. Uropathogenic Escherichia coli, most causative agent of Urinary tract infection, has developed resistance to various antibiotics. In the present investigation, antibacterial substance like bacteriocin (Bacteriocin DT24) produced by probiotic Lactobacillus brevis DT24 from vaginal sample of healthy Indian woman was partially purified and characterized. It was efficiently working against various pathogens, that is, Uropathogenic E. coli, Enterococcus faecium, Enterococcus faecalis and Staphylococcus aureus. The antimicrobial peptide was relatively heat resistant and also active over a broad range of pH 2–10. It has been partially purified by ammonium sulfate precipitation and gel filtration chromatography and checked on reverse-phase high-performance liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of bacteriocin DT24 was approximately 7-kDa protein. The peptide is inactivated by proteolytic enzymes, trypsin and lipase but not when treated with catalase, α-amylase and pepsin. It showed bacteriostatic mode of action against uropathogenic E. coli. Such characteristics indicate that this bacteriocin-producing probiotic may be a potential candidate for alternative agents to control urinary tract infections and other pathogens.     

Pseudomonas putida Strain FStm2 Isolated from Shark Skin: A Potential Source of Bacteriocin

Bacteriocin-producing Pseudomonas putida strain FStm2 isolated from shark showed broad range of antibacterial activity against all pathogens tested except Bacillus subtilis ATCC11774, MRSA N32064, Proteus mirabilis ATCC12453, Enterococcus faecalis ATCC14506, Salmonella typhimurium ATCC51312, Salmonella mutan ATCC25175, and Aeromonas hydrophila Wbf314. Of the three growth media tested in this study, TSB was observed to support the bacteriocin activity the most. While the highest bacteriocin activity was observed for media supplemented with 1 % NaCl, there was an observed reduction in bacteriocin activity with increasing salt concentration. Although the least bacteriocin activity was observed for marine broth, addition of increasing amounts of tryptone, glucose, or yeast extract increased bacteriocin activity. This was, however, contrary to the effect observed when MgSO₄ and MnSO₄ were added as supplements. In the presence of α-amylase, lipase, DNase, and RNase, a positive effect on bacteriocin production was observed. Proteinase K strongly inhibited bacteriocin production. Furthermore, the bacteriocins produced were heat stable within the temperature range of 30–70 °C. Bacteriocin activity also was not affected within a wide pH range of 3–9. Exposure to detergents did not inhibit the activity of the bacteriocin at the concentrations tested. Instead, a positive effect on the relative activity of produced bacteriocin was observed as sodium dodecyl sulfate (SDS), EDTA, and Tween 20 at 1 % concentration all improved bacteriocin activity when the cell-free supernatant was tested against Serratia marcescens ATCC 13880. The bacteriocin was purified by ammonium sulfate precipitation and gel filtration on a Superdex-200 column. SDS-PAGE analysis of the partially purified bacteriocin revealed an apparent molecular weight of ~32 kDa.     

The inhibition of fungal cellulolysis by cell-free preparations from ruminococci

The degradation of filter paper by the anaerobic fungus Neocallimastix frontalis strain RE1 was reduced by the addition of cell-free supernates from cultures of Ruminococcus albus strain J6 and R. flavefaciens strains 17 and 007. Fungal uptake of, and growth on, glucose was not affected. After gel permeation and anion exchange chromatography, inhibitory activity towards fungal cellulolysis was recovered in a fraction from strain 17 that contained at least five negatively charged polypeptide components, molecular mass 45-68 kDa, on SDS-PAGE.     

Restriction of a bacteriophage of Streptomyces albus G involving endonuclease SalI.

The bacteriophage Pa16, isolated from soil on Streptomyces albus G, was restricted when transferred from an alternative host back to S. albus G. Extracted unmodified Pa16 deoxyribonucleic acid was cleaved at a single site by a cell-free extract of S. albus G. Fractions cleaving Pal6 deoxyribonucleic acid contained the endonuclease SalI first described by J. Arrand, P. Myers, and R. J. Roberts (unpublished data). A mutant of S. albus G was isolated which was defective in both restriction and modification of Pal6. This mutant lacked SalI activity. It is concluded that SalI is the agent of restriction of Pal6 by S. albus G.     

Purification and partial characterization of a bacteriocin produced by Eikenella corrodens

Aims: The purpose of this study was to purify and characterize a bacteriocin produced by Eikenella corrodens A32E2. Methods and Results: Peptostreptococcus anaerobius ATCC27337 was used as indicator strain in antagonistic assays for bacteriocin‐producing E. corrodens A32E2. Protein extraction was influenced by pH and buffer composition. The protein was active in the pH range 6–8. Inhibitory activity was lost by both heating and treatment with proteolytic enzymes and decreased with organic solvents. The substance is rather unstable but maintains 100% of its activity after being exposed to acetone and when stored at ?70°C. The antagonistic substance was first precipitated by ammonium sulfate and further partially purified by Mono‐Q FPLC and C‐18 HPLC. Mass spectrometry analysis showed that the molecular mass was 23 625 Da, and the sequence obtained for the N‐terminus was: Met‐Asn‐Phe‐Asp‐Glu‐Lys‐Val‐Gly‐Lys‐Val‐X‐Phe‐Lys‐Val‐Gly‐Asp. Conclusions: The evidence presented in this study supports the idea that an antagonistic substance produced by E. corrodens A32E2 isolated from a periodontal diseased site is a novel bacteriocin, which we designate corrodecin. Significance and Impact of the Study:  We anticipated that corrodecin might play an important role at the periodontal site. This compound could also be attractive in biotechnological applications as an interesting tool for oral ecosystem control.     

Purification and characterization of a bacteriocin from Lactobacillus rhamnosus L34

Bacteriocins are ribosomally synthesized peptides having considerable potential as a food preservative because of their strong antagonistic activity against many food spoilage and pathogenic organisms. A bacteriocin from Lactobacillus rhamnosus isolates was purified using ammonium sulphate precipitation and molecular exclusion chromatography techniques. Ammonium sulphate precipitation resulted in higher yield of bacteriocin, but the specific activity and fold purification were higher for molecular exclusion chromatography. The bacteriocin exhibited inhibition against food-borne pathogens and spoilage microorganisms, including both Gram-positive and -negative bacteria. Amylase, lipase and catalase did not alter the antimicrobial activity but proteolytic enzymes inactivated the bacteriocin. It was heat stable and exhibited activity in a pH range of 2–8 with maximum activity at pH 5.0. Molecular weight of bacteriocin was found to be ~5.6 kDa using SDS-PAGE. HPLC profile showed a single peak further attesting the purity of the bacteriocin.     

Production and partial purification of a peptide antibiotic from Staphylococcus epidermidis

When grown on solid or in liquid Brain Heart Infusion at 37°C, Staphylococcus epidermidis NCIB 11536 produced antibiotic activity against a wide range of Gram positive bacteria. Production was influenced by aeration, pH, glucose concentration and specific growth rate. Inhibitory activity could be concentrated by ammonium sulphate precipitation (30–55% saturation). On Sephadex G50 using 0.05 mol/1 sodium phosphate buffer, pH 6.0, two peaks of antibiotic activity were detected. The first peak eluted with the void volume (K_d= 0) and the second peak was retained by the gel (K_d= 0.73–0.77). These two substances did not represent the monomeric and polymeric forms of a staphylococcal bacteriocin. The low mol. wt inhibitor, which was responsible for over 95% of the recovered activity on Sephadex G50, could be partially purified by a combination of gel filtration on Biogel P2 and ion-exchange chromatography on Sephadex C-25. Yields were increased by combining these two steps into a single procedure (duocolumn). The semi-purified inhibitor was desalted using Sep-pak C₁₈ cartridges. Biological activity was resistant to enzymic denaturation except by high concentrations of trypsin (50 units/μg, 3 h, 25°C). This peptide antibiotic is different from any previously described staphylococcal inhibitors.