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L-forms induced from a bacteriocin-producing strain of group A streptococcus retained both the ability to produce the streptococcin and producer strain immunity to the homologous bacteriocin. L-forms of a spontaneously cured (bacteriocin negative) derivative of this same strain failed to produce streptococcin but were sensitive to its action.
The bacteriocin and hemolysin produced by a Streptococcus faecium strain were partially separated after gel filtration on Ultrogel AcA-22. The molecular weight of bacteriocin was approximately 300,000, and that of hemolysin was 220,000. Non-bacteriocinogenic mutants retained the hemolytic activity. Production of hemolysin required glucose, whereas that of bacteriocin did not.
Syringacin 4-A, a bacteriocin produced by Pseudomonas syrinagae 4-A, was obtained by induction with ultraviolet irradiation or mitomycin C. Approximately 1,000-fold purification of the bacteriocin was achieved by manganous chloride precipitation, differential centrifugation, and chromatography on hydroxyapatite columns. The purified syngacin was homogeneous on hydroxyapatite columns and sucrose density gradients; it also sedimented as a single entity in the analytical ultracentrifuge. The buoyant density of purified syringacin in cesium chloride was 1.294 g/ml. The sedimentation coefficient was calculated as 120S, and the diffusion coefficient was 6.49 x 10(-8) cm(2)/s. The molecular weight was calculated as 1.6 x 10(7) from physical data and 1.7 x 10(7) from biological data. The syringacin was composed of about 88.4% protein, 8.5% arabinose, 2.2% galacturonic acid, and 0.7% glucosamine. Amino acid analysis indicated a predominance of leucine (12.1%), aspartic acid (12.2%), and glutamic acid (12.7%). The ultraviolet spectrum showed a maximum absorbance peak at 276 nm. The syringacin was heat and alcohol sensitive, but resistant to trypsin, chymotrypsin, carboxypeptidase, Pronase, protease, lysozyme, steapsin, deoxyribonuclease, and ribonuclease. Maximum pH stability was between 5 and 8. Crude bacteriocin was stable at room temperature for at least a year, and purified material was stable for at least 3 months at 4 C.
Bacteriocin-like activities were detected in 78% of 120 alpha-hemolytic streptococcal isolates. Inhibitory substances from three such isolates (one Streptococcus sanguis strain and two S. mitis strains) were investigated further and termed viridins (A, B, and C). The viridins were unique among bacteriocins of gram-positive bacteria in that they inhibited many gram-negative bacteria in addition to inhibition of a variety of gram-positive organisms. Viridins were obtained in a cell-free state only after mechanical disruption of bacteriocinogenic cells but could not be isolated from supernatant fluids of cultures of such bacteria or from freeze-thaw liquor of agar on which the bacteria had been grown. Viridin B could be partially purified by ammonium sulfate precipitation and by gel filtration on a Sephadex G-100 column. This bacteriocin had some unusual properties including heat lability, a narrow pH range of activity, and lack of adsorptive capacity to susceptible bacteria. Although viridin B was bactericidal to a Neisseria sicca strain, it was only bacteriostatic against a coagulase-negative staphylococcus.
The locations of the genetic determinants for enterocin L50 (EntL50A and EntL50B), enterocin Q (EntQ), and enterocin P (EntP) in the multiple bacteriocin producer Enterococcus faecium strain L50 were determined. These bacteriocin genes occur at different locations; entL50AB (encoding EntL50A and EntL50B) are on the 50-kb plasmid pCIZ1, entqA (encoding EntQ) is on the 7.4-kb plasmid pCIZ2, and entP (encoding EntP) is on the chromosome. The complete nucleotide sequence of pCIZ2 was determined to be 7,383 bp long and contains 10 putative open reading frames (ORFs) organized in three distinct regions. The first region contains three ORFs: entqA preceded by two divergently oriented genes, entqB and entqC. EntqB shows high levels of similarity to bacterial ATP-binding cassette (ABC) transporters, while EntqC displays no significant similarity to any known protein. The second region encompasses four ORFs (orf4 to orf7), and ORF4 and ORF5 display high levels of similarity to mobilization proteins from E. faecium and Enterococcus faecalis. In addition, features resembling a transfer origin region (oriT) were found in the promoter area of orf4. The third region contains three ORFs (orf8 to orf10), and ORF8 and ORF9 exhibit similarity to the replication initiator protein RepE from E. faecalis and to RepB proteins, respectively. To clarify the minimum requirement for EntQ synthesis, we subcloned and heterologously expressed a 2,371-bp fragment from pCIZ2 that encompasses only the entqA, entqB, and entqC genes in Lactobacillus sakei, and we demonstrated that this fragment is sufficient for EntQ production. Moreover, we also obtained experimental results indicating that EntqB is involved in ABC transporter-mediated EntQ secretion, while EntqC confers immunity to this bacteriocin.
Growth of and bacteriocin production by Streptococcus macedonicus ACA-DC 198 were assessed and modeled under conditions simulating Kasseri cheese production. Controlled fermentations were performed in milk supplemented with yeast extract at different combinations of temperature (25, 40, and 55 degrees C), constant pH (pHs 5 and 6), and added NaCl (at concentrations of 0, 2, and 4%, wt/vol). The data obtained were used to construct two types of predictive models, namely, a modeling approach based on the gamma concept, as well as a model based on artificial neural networks (ANNs). The latter computational methods were used on 36 control fermentations to quantify the complex relationships between the conditions applied (temperature, pH, and NaCl) and population behavior and to calculate the associated biokinetic parameters, i.e., maximum specific growth and cell count decrease rates and specific bacteriocin production. The functions obtained were able to estimate these biokinetic parameters for four validation fermentation experiments and obtained good agreement between modeled and experimental values. Overall, these experiments show that both methods can be successfully used to unravel complex kinetic patterns within biological data of this kind and to predict population kinetics. Whereas ANNs yield a better correlation between experimental and predicted results, the gamma-concept-based model is more suitable for biological interpretation. Also, while the gamma-concept-based model has not been designed for modeling of other biokinetic parameters than the specific growth rate, ANNs are able to deal with any parameter of relevance, including specific bacteriocin production.
A total of 52 strains of Lactobacillus acidophilus were examined for production of bacteriocins. A majority (63%) demonstrated inhibitory activity against all members of a four-species grouping of Lactobacillus leichmannii, Lactobacillus bulgaricus, Lactobacillus helveticus, and Lactobacillus lactis. Four L. acidophilus strains with this activity also inhibited Streptococcus faecalis and Lactobacillus fermentum, suggesting a second system of antagonism. Under conditions eliminating the effects of organic acids and hydrogen peroxide, no inhibition of other gram-positive or -negative genera was demonstrated by L. acidophilus. The agent produced by L. acidophilus N2 and responsible for inhibition of L. leichmannii, L. bulgaricus, L. helveticus, and L. lactis was investigated. Ultrafiltration studies indicated a molecular weight of approximately 100,000 for the crude inhibitor. The agent was sensitive to proteolytic enzymes and retained full activity after 60 min at 100 degrees C (pH 5). Activity against sensitive cells was bactericidal but not bacteriolytic. These characteristics identified the inhibitory agent as a bacteriocin, designated lactacin B. Examination of strains of L. acidophilus within the six homology groupings of Johnson et al. (Int. J. Syst. Bacteriol. 30:53-68, 1980) demonstrated that production of the bacteriocin lactacin B could not be used in classification of neotype L. acidophilus strains. However, the usefulness of employing sensitivity to lactacin B in classification of dairy lactobacilli is suggested.
The mechanism of bactericidal activity of lactostrepcin 5 (Las 5), a bacteriocin produced by Streptococcus cremoris 202, was investigated. Las 5 did not kill protoplasts of sensitive cells, and its activity was decreased about 10-fold after pretreatment of the cells with trypsin, suggesting the involvement of the cell wall in the activity of this bacteriocin. In susceptible cells, the bacteriocin slowed down and then stopped synthesis of DNA, RNA, and protein, although this did not appear to be the primary effect of Las 5 action. Las 5 also inhibited uridine transport in susceptible cells and induced leakage of K+ ions and ATP. Survival of cells treated with Las 5 in phosphate buffer was higher in the presence of K+, CA2+, or Mg2+ ions.
The bacteriocin produced by Pediococcus acidilactici PAC 1.0, previously designated PA-1 bacteriocin, was found to be inhibitory and bactericidal for Listeria monocytogenes. A dried powder prepared from PAC 1.0 culture supernatant fortified with 10% milk powder was found to contain bacteriocin activity. An MIC against L. monocytogenes and lytic effects in broth cultures were determined. Inhibition by PA-1 powder occurred over the pH range 5.5 to 7.0 and at both 4 and 32 degrees C. In addition, inhibition of L. monocytogenes was demonstrated in several food systems including dressed cottage cheese, half-and-half cream, and cheese sauce.
Lactacin F, a bacteriocin produced by Lactobacillus acidophilus 11088 (NCK88), was purified and characterized. Lactacin F is heat stable, proteinaceous, and inhibitory to other lactobacilli as well as Enterococcus faecalis. The bacteriocin was isolated as a floating pellet from culture supernatants brought to 35 to 40% saturation with ammonium sulfate. Native lactacin F was sized at approximately 180 kDa by gel filtration. Column fractions having lactacin F activity were examined by electron microscopy and contained micelle-like globular particles. Purification by ammonium sulfate precipitation, gel filtration, and high-performance liquid chromatography resulted in a 474-fold increase in specific activity of lactacin F. The purified bacteriocin was identified as a 2.5-kDa peptide by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The lactacin F peptide retained activity after extraction from SDS-PAGE gel slices, confirming the identity of the 2.5-kDa peptide. Variants of NCK88 that failed to exhibit lactacin F activity did not produce the 2.5-kDa band. Sequence analysis of purified lactacin F identified 25 N-terminal amino acids containing an arginine residue at the N terminus. Composition analysis indicates that lactacin F may contain as many as 56 amino acid residues.
Ten strains of bacteriocin-producing lactic acid bacteria were isolated from retail cuts of meat. These 10 strains along with 11 other bacteriocin-producing lactic acid bacteria were tested for inhibitory activity against psychotrophic pathogens, including four strains of Listeria monocytogenes, two strains of Aeromonas hydrophila, and two strains of Staphylococcus aureus. Inhibition due to acid, hydrogen peroxide, and lytic bacteriophage were excluded. The proteinaceous nature of the inhibitory substance was confirmed by demonstration of its sensitivity to proteolytic enzymes. Eight of the meat isolates had inhibitory activity against all four L. monocytogenes strains. Bacteriocin activity against L. monocytogenes was found in all of the strains obtained from other sources. Activity against A. hydrophila and S. aureus was also common.
A bovine intestinal bacterial isolate, identified as Enterococcus hirae, was found to produce a bacteriocin (designated hiraecin S) inhibitory to Listeria monocytogenes and other Listeria spp. Identification to species level was determined by comprehensive biochemical and morphological tests which were verified by DNA-DNA homology assays. The antimicrobial agent was inactivated by pronase and papain and was insensitive to catalase. The antimicrobial activity was not due to hydrogen peroxide or acid formation, nor was lysozyme or muramidase activity observed in cell-free bacteriocin preparations. Inhibition of selected gram-negative bacteria was not observed. Other enterococci were sensitive to the bacteriocin, and except for Listeria spp., no other gram-positive bacteria tested were inhibited.
A bacteriocin-producing Carnobacterium sp. was isolated from fish. The bacteriocin, termed carnocin UI49, was purified to homogeneity by a four-step purification procedure, including hydrophobic interaction chromatography and reverse-phase chromatography. Carnocin UI49 has a bactericidal mode of action. It was shown to be heat tolerant and stable between pH 2 and 8. At pH above 8, carnocin UI49 was rapidly inactivated. Amino acid analysis revealed a composition of about 35 to 37 amino acids in addition to an unidentified peak which migrates at the position of lanthionine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis suggests a molecular weight of about 4,500 to 5,000. Mass spectrometry gave a molecular weight of 4,635, which is about 1,000 larger than that calculated from the amino acid analysis data. Performic acid oxidation of carnocin UI49, followed by amino acid hydrolysis, revealed the presence of cysteic acid. The sequence of the first seven amino acid residues was determined to be N-Gly-Ser-Glu-Ile-Gln-Pro-Arg. After the seventh amino acid, carnocin UI49 was not available for further Edman degradation. The results suggest that carnocin UI49 belongs to the class of bacteriocins termed lantibiotics.
Production of propionicin PLG-1 by Propionibacterium thoenii P127 was pH dependent, with maximal activity detected in supernatants of cultures grown at pH 7.0 Propionicin PLG-1 was purified by ion-exchange chromatography and isoelectric focusing. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of propionicin PLG-1 purified through isoelectric focusing resolved a protein band with a molecular weight of 10,000. Propionicin PLG-1 was bactericidal to sensitive cells, demonstrating single-hit kinetics. The producing strain harbored a single plasmid (pLG1) with an approximate size of 250 kb. Preliminary data indicate that both propionicin PLG-1 and immunity to the bacteriocin are encoded on the chromosome. Exposure of strain P127 to acriflavine or to N-methyl-N'-nitro-N-nitrosoguanidine yielded isolates that no longer produced bacteriocin activity and isolates that were cured of the plasmid. However, loss of bacteriocin production was not correlated with loss of the plasmid. Isolates cured of the plasmid were phenotypically identical to plasmid-bearing cells in fermentation patterns, pigment production, and growth characteristics.
Purification and amino acid sequencing of plantaricin A, a bacteriocin from Lactobacillus plantarum C11, revealed that maximum bacteriocin activity is associated with the complementary action of two almost-identical peptides, alpha and beta (J. Nissen-Meyer, A. G. Larsen, K. Sletten, M. Daeschel, and I. F. Nes, J. Gen. Microbiol. 139:1973-1978, 1993). A 5-kb chromosomal HindIII restriction fragment containing the structural gene of plantaricin A was cloned and sequenced. Only one gene encoding plantaricin A was found. The gene, termed plnA, encodes a 48-amino-acid precursor peptide, of which the 22 and 23 C-terminal amino acids correspond to the purified peptides. Northern (RNA) blot analysis demonstrated that a probe complementary to the coding strand of the plantaricin A gene hybridized to a 3.3-kb mRNA transcript. Further analysis of the 3.3-kb transcript demonstrated that it contains three additional open reading frames (plnB, plnC and plnD) downstream of plnA. The DNA sequences of plnB, plnC, and plnD revealed that their products closely resemble members of bacterial two-component signal transduction systems. The strongest homology was found to the accessory gene regulatory (agr) system, which controls expression of exoproteins during post-exponential growth in Staphylococcus aureus. The finding that plnABCD are transcribed from a common promoter suggests that the biological role played by the bacteriocin is somehow related to the regulatory function of the two-component system located on the same operon.
Curvaticin FS47, a bacteriocin produced by Lactobacillus curvatus FS47, is inhibitory to Listeria monocytogenes, as well as Lactobacillus, Pediococcus, Enterococcus, and Bacillus spp. The bacteriocin was purified by 40% ammonium sulfate precipitation, solid-phase extraction, and reversed-phase high-pressure liquid chromatography. Purified curvaticin FS47 was determined to be 4.07 kDa by mass spectrometry and was partially sequenced. Thirty-one N-terminal amino acids were identified; the curvaticin FS47 protein sequence did not show homology to the pediocin-like group of bacteriocins.
Leucocin A is a small heat-stable bacteriocin produced by Leuconostoc gelidum UAL187. A 2.9-kb fragment of plasmid DNA that contains the leucocin structural gene and a second open reading frame (ORF) in an operon was previously cloned (J. W. Hastings, M. Sailer, K. Johnson, K. L. Roy, J. C. Vederas, and M. E. Stiles, J. Bacteriol. 173:7491-7500, 1991). When a 1-kb DraI-HpaI fragment containing this operon was introduced into a bacteriocin-negative variant (UAL187-13), immunity but no leucocin production was detected. Leucocin production was observed when an 8-kb SacI-HindIII fragment of the leucocin plasmid was introduced into L. gelidum UAL187-13 and Lactococcus lactis IL1403. Nucleotide sequence analysis of this 8-kb fragment revealed the presence of three ORFs in an operon upstream of and on the strand opposite from the leucocin structural gene. The first ORF (lcaE) encodes a putative protein of 149 amino acids with no apparent function in leucocin A production. The second ORF (lcaC) contains 717 codons that encode a protein homologous to members of the HlyB family of ATP-binding cassette transporters. The third ORF (lcaD) contains 457 codons that encode a protein with marked similarity to LcnD, a protein essential for the expression of the lactococcal bacteriocin lactococcin A. Deletion mutations in lcaC and lcaD resulted in loss of leucocin production, indicating that LcaC and LcaD are involved in production and translocation of leucocin A. The secretion apparatus for lactococcin A did not complement mutations in the lcaCD genes to express leucocin A in L. lactis.(ABSTRACT TRUNCATED AT 250 WORDS)
Two bacteriocins produced by Carnobacterium piscicola V1 were purified and characterized. Piscicocin V1a (molecular mass = 4,416 Da) and piscicocin V1b (molecular mass = 4,526 Da) are nonlantibiotic, small, heat-stable antibacterial peptides. Piscicocin V1b is identical to carnobacteriocin BM1, while piscicocin V1a is a new bacteriocin. Its complete sequence of 44 amino acid residues has been determined. Piscicocin V1a belongs to the class IIa bacteriocins having the consensus YGNGV motif. These peptides inhibit various gram-positive bacteria, including Listeria monocytogenes. Piscicocin V1a is approximately 100 times more active than piscicocin V1b against indicator strains. However, the antagonistic spectrum is the same for both piscicocins. Comparison of these results with the analysis of the amino acid sequence and secondary structure predictions suggests that (i) the conserved N-terminal conserved domain is involved in the receptor recognition and therefore in an "all-or-none" response against target bacterial cells and (ii) the C-terminal variable and hydrophobic domain determines membrane anchoring and therefore the intensity of the antagonist response.