Takatoshi Itoh

Phosphate group requirement for mitogenic activation of lymphocytes by an extracellular phosphopolysaccharide from Lactobacillus delbrueckii ssp. bulgaricus

The mitogenic activity of extracellular polysaccharides produced by Lactobacillus delbrueckii ssp. bulgaricus OLL 1073R-1 and NCFB2483 was examined in murine lymphocytes. The extracellular polysaccharide from Lactobacillus delbrueckii ssp. bulgaricus OLL 1073R-1 was fractionated into neutral and acidic polysaccharides by anion-exchange chromatography, while that of Lactobacillus delbrueckii ssp. bulgaricus NCFB2483 were all fractionated into neutral polysaccharide(s). The acidic polysaccharide stimulated mitogenic responses of murine splenocytes and Peyers patches but not of thymocytes. The optimal concentration of the acidic polysaccharide at the highest stimulation was 160 microg/ml. A significant increase of mitogenic activity was initiated at 24 h, and the highest response was obtained after stimulation for 48 h. The acidic polysaccharide purified by high performance liquid chromatography also had substantial mitogenic activity, and the molecular weight was estimated to be 1.2 x 10(6). The acidic polysaccharide was a phosphopolysaccharide consisting of glucose, galactose and phosphorus. Dephosphorylation by hydrofluoric acid degradation reduced the mitogenic activity in lymphocytes. The phosphopolysaccharide produced by Lactobacillus delbrueckii ssp. bulgaricus 1073R-1 is a potent B-cell-dependent mitogen in which the phosphate group acts as a trigger of the mitogenic induction.

Structure of the extracellular polysaccharide from slime-forming Lactococcus lactis subsp. cremoris SBT 0495

The extracellular polysaccharide obtained from slime-forming Lactococcus lactis subsp. cremoris SBT 0495 is composed of D-glucose, D-galactose, L-rhamnose, and phosphate. Methylation analysis of the native and dephosphorylated polysaccharides provided information on the linkage of the sugar residues and the location of the phosphate group. N.m.r. spectroscopy confirmed the structure of the polysaccharide, which is assigned the following repeating-unit: [formula: see text]

Inhibition of food‐borne pathogenic bacteria by bacteriocins from Lactobacillus gasseri

Seventy‐three strains of the Lactobacillus acidophilus group and a Lact. reuteri isolated from human faeces were examined for production of antimicrobial agents against 16 strains of six species of food‐borne enteric pathogenic bacteria. Several strains of Lact. gasseri showed wide inhibitory activity against the tested bacteria. Gassericin A produced by Lact. gasseri LA39 was one of the most widely active bacteriocins. It was bactericidal without causing cell lysis.

Structural and functional differences in two cyclic bacteriocins with the same sequences produced by lactobacilli

ABSTRACT Lactobacillus gasseri LA39 and L. reuteri LA6 isolated from feces of the same human infant were found to produce similar cyclic bacteriocins (named gassericin A and reutericin 6, respectively) that cannot be distinguished by molecular weights or primary amino acid sequences. However, reutericin 6 has a narrower spectrum than gassericin A. In this study, gassericin A inhibited the growth of L. reuteri LA6, but reutericin 6 did not inhibit the growth of L. gasseri LA39. Both bacteriocins caused potassium ion efflux from indicator cells and liposomes, but the amounts of efflux and patterns of action were different. Although circular dichroism spectra of purified bacteriocins revealed that both antibacterial peptides are composed mainly of α-helices, the spectra of the bacteriocins did not coincide. The results of d- and l-amino acid composition analysis showed that two residues and one residue of d-Ala were detected among 18 Ala residues of gassericin A and reutericin 6, respectively. These findings suggest that the different d-alanine contents of the bacteriocins may cause the differences in modes of action, amounts of potassium ion efflux, and secondary structures. This is the first report that characteristics of native bacteriocins produced by wild lactobacillus strains having the same structural genes are influenced by a difference in d-amino acid contents in the molecules.

Acidophilucin A, a new heat-labile bacteriocin produced by Lactobacillus acidophilus LAPT 1060

Lactobacillus acidophilus LAPT 1060, isolated from infant faeces, produced an antimicrobial agent active against six strains of Lactobacillus delbrueckii subsp. bulgaricus and six strains of Lactobacillus helveticus. The agent was sensitive to proteolytic enzymes and heat (10 min at 60°C) and is a bacteriocin and designated acidophilucin A.

Reutericin 6, a new bacteriocin produced by Lactobacillus reuteri LA 6

Lactobacillus reuteri LA 6, isolated from infant faeces, produced an antimicrobial agent active against Lactobacillus acidophilus JCM 2125, Lactobacillus delbrueckii subsp. bulgaricus JCM 1002 and Lactobacillus delbrueckii subsp. lactis JCM 1148 and JCM 1248. The agent was sensitive to proteolytic enzymes and retained activity after heating at 100°C for 20 min. This agent was a bacteriocin and has been designated as reutericin 6. Reutericin 6 exceeds 200 kDa as determined by ultrafiltration studies. Activity against sensitive cells was both bacteriocidal and bacteriolytic.

Lacticin, a bacteriocin produced by Lactobacillus delbrueckii subsp. lactis

Twenty‐one strains of Lactobacillus delbrueckii and L. helveticus were tested for bacteriocin production against each other. Lactobacillus delbrueckii subsp. lactis JCM 1106 and 1107 produced an inhibitory agent active against L. delbrueckii subsp. bulgaricus JCM 1002 and NIAI yB‐62, L. delbrueckii subsp. lactis JCM 1248 and L. delbrueckii subsp. delbrueckii JCM 1012. Lactobacillus delbrueckii subsp. lactis JCM 1248 inhibited only the growth of L. delbrueckii subsp. bulgaricus NIAI yB‐62. These agents were sensitive to proteolytic enzymes and heating (at 60°C for 10min). These agents were considered to be bacteriocins and designated lacticin A and B.

Potential of Lactobacillus gasseri isolated from infant faeces to produce bacteriocin

Thirty strains of Lactobacillus gasseri isolated from infant faeces were examined for production of an antimicrobial agent against 10 strains of the seven species of the genus Lactobacillus. Each of the strains inhibited the growth of at least one of the indicator strains. The agent was sensitive to proteolytic enzymes and stable for 20 min at 120°C. It is a bacteriocin designated as gassericin A.

Primary amino acid and DNA sequences of gassericin T, a lactacin F-family bacteriocin produced by Lactobacillus gasseri SBT2055.

A broad-spectral bacteriocin, named gassericin T, produced by Lactobacillus gasseri SBT 2055 (from human feces) was isolated to homogeneity from the culture supernatant by hydrophobic chromatography. By SDS-PAGE and in situ activity assay, the purified gassericin T migrated as a single band with bacteriocin activity and molecular size of 5,400. A 2.9-kbp HindIII-HindIII fragment of chromosome DNA was hybridized with the oligonucleotide probe designed from the partial N-terminal amino acid sequence of gassericin T and was cloned. Six ORFs including the structural gene of gassericin T were deduced by computer analysis and the data bases. The structural gene of gassericin T (gatA) was identified as the fourth ORF, which encoded a protein composed of 75 amino acids that included the GG motif of the cleavage site. Chemical sequencing analysis of the complete amino acid sequence showed that gassericin T (57 amino acids) had a disulfide bond in the molecule and no modified amino acid residues, making it a class II bacteriocin. The gassericin T had 60% sequence similarity to mature LafA (57 amino acids, lactacin F, bacteriocins produced by L. johnsonii VPI11088), and the sequences around the processing site and C-terminal area were well conserved. The fifth ORF was designated as gatX, encoded as a peptide composed of 65 amino acids containing the GG motif of the putative cleavage site, however mature GatX and its antibacterial activity were not detected in the culture supernatant. GatX has higher similarity with LafX than with lactobin A (50 amino acids) belonging to the first lactacin F-family. These results indicated that gassericin T belongs to the hydrophobic class II bacteriocins and the most vicinal lactacin F-family.

Presence of two neutral disaccharides containing N-acetylhexosamine in bovine colostrum as free forms

Two neutral disaccharides which comprise 74.0% of the neutral oligosaccharides other than lactose were isolated from bovine colostrum taken 6 h after parturition. The chemical structures were revealed to be galactosyl-beta-1,4-N-acetylglucosamine (N-acetyllactosamine, 70.3%) and N-acetylgalactosaminyl-beta-1,4-glucose (3.7%). The two carbohydrates were the newly found oligosaccharides from mammalian milk in the free forms. 7 days after parturition, they had completely disappeared from bovine milk.