Isolation and Partial Amino Add Sequence of Bacteriocins Produced by Lactobacillus acidophilus

Bacteriocins produced by Lactobacillus acidophilus JCM 1023, JCM 1028, JCM 1021, JCM 1229, and JCM 5342 were active against closely related lactobacilli. These bacteriocins were purified and partial sequenced. Bacteriocin activities of L. acidophilus JCM 1023 and JCM 1028 were associated with two components. On the basis of N-terminal amino acid sequencing and the molecular masses, it is interpreted that these two-component bacteriocins are identical to acidocin J1132, a bacteriocin from L. acidophilus JCM 1132 [Tahara et al., Appl. Environ. Microbiol., 62, 892–897 (1996)]. Other bacteriocins were single-peptide bacteriocins.     

Detection and quantification of probiotic strain Lactobacillus gasseri K7 in faecal samples by targeting bacteriocin genes

Lactobacillus gasseri K7 is a probiotic strain that produces bacteriocins gassericin K7 A and K7 B. In order to develop a real-time quantitative PCR assay for the detection of L. gasseri K7, 18 reference strains of the Lactobacillus acidophilus group and 45 faecal samples of adults who have never consumed strain K7 were tested with PCR using 14 pairs of primers specific for gassericin K7 A and K7 B gene determinants. Incomplete gassericin K7 A or K7 B gene clusters were found to be dispersed in different lactobacilli strains as well as in faecal microbiota. One pair of primers was found to be specific for the total gene cluster of gassericin K7A and one for gassericin K7B. The real-time PCR analysis of faecal samples spiked with K7 strain revealed that primers specific for the gene cluster of the gassericin K7 A were more suitable for quantitative determination than those for gassericin K7 B, due to the lower detection level. Targeting of the gassericin K7 A or K7 B gene cluster with specific primers could be used for detection and quantification of L. gasseri K7 in human faecal samples without prior cultivation. The results of this study also present new insights into the prevalence of bacteriocin-encoding genes in gastrointestinal tract.     

Characterization and Cytotoxic Evaluation of Bacteriocins Possessing Antibiofilm Activity Produced by Lactobacillus plantarum SJ33

International Journal of Peptide Research and Therapeutics - Biofilm forming pathogens are among the major causes of hospital-acquired infections and are not much affected by antibiotic treatment....     

High-Frequency Plasmid Transduction by Lactobacillus gasseri Bacteriophage phiadh

The temperate bacteriophage phiadh mediates plasmid DNA transduction in Lactobacillus gasseri ADH at frequencies in the range of 10 to 10 transductants per PFU. BglII-generated DNA fragments from phage phiadh were cloned into the BclI site of the transducible plasmid vector pGK12 (4.4 kb). Phage phiadh lysates induced from Lactobacillus lysogens harboring pGK12 or the recombinant plasmids were used to transduce strain ADH to chloramphenicol resistance. The transduction frequencies of recombinant plasmids were 10- to 10-fold higher than that of native pGK12. The increase in frequency generally correlated with the extent of DNA-DNA homology between plasmid and phage DNAs. The highest transduction frequency was obtained with plasmid pTRK170 (6.6 kb), a pGK12 derivative containing the 1.4- and 0.8-kb BglII DNA fragments of phiadh. DNA hybridization analysis of pTRK170-transducing phage particles revealed that pTRK170 had integrated into the phiadh genome, suggesting that recombination between homologous sequences present in phage and plasmid DNAs was responsible for the formation of high-frequency transducing phage particles. Plasmid DNA analysis of 13 transductants containing pTRK170 showed that each had acquired intact plasmids, indicating that in the process of transduction a further recombination step was involved in the resolution of plasmid DNA monomers from the recombinant pTRK170::phiadh molecule. In addition to strain ADH, pTRK170 could be transduced via phiadh to eight different L. gasseri strains, including the neotype strain, F. Gasser 63 AM (ATCC 33323).     

Isolation and characterization of a new bacteriocin from Lactobacillus gasseri KT7

A bacteriocin-producing Lactobacillus gasseri strain, KT7, was isolated from infant faeces. The supernatant fluid showed inhibitory activity not only against some lactic acid bacteria but also, against some pathogenic and food-spoilage species, including Clostridium, Listeria and Enterococcus. An antimicrobial peptide designated gassericin KT7 was isolated from Lactobacillus gasseri KT7. It was purified to homogeneity by a single four-step procedure: a crude supernatant fluid obtained from early stationary-phase culture in MRS medium was subjected to ammonium sulphate fractionation, CM-Sephadex cation-exchange chromatography, Phenyl-Sepharose hydrophobic chromatography and reverse-phase HPLC chromatography. Gassericin KT7 was sensitive to proteolytic enzymes, resistant to heat, active over a wide range of pH, and migrated as a 4.5-5.0 kDa peptide on SDS-PAGE. The bacteriocin was produced constitutively during exponential growth. It was bactericidal to sensitive cells and the bactericidal effect was not produced by cell lysis. The amino acid composition of the bacteriocin was determined and no modified amino acid was found among the residues identified.     

Salivaricin P, One of a Family of Two-Component Antilisterial Bacteriocins Produced by Intestinal Isolates of Lactobacillus salivarius

Lactobacillus salivarius DPC6005, a porcine intestinal isolate, produces a two-component bacteriocin, salivaricin P, with homology to ABP-118 produced by a human probiotic L. salivarius strain. Indeed, molecular characterization revealed that while the peptides Sln1 and ABP-118α are identical, their companion peptides (Sln2 and ABP-118β, respectively) differ by two amino acids. This observation suggests that two-component bacteriocins may be a common feature of intestinal L. salivarius strains.     

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.     

Evaluation of the passage of Lactobacillus gasseri K7 and bifidobacteria from the stomach to intestines using a single reactor model

Background

The antibacterial activity of bacteriophages has been described rather well. However, knowledge about the direct interactions of bacteriophages with mammalian organisms and their other, i.e. non-antibacterial, activities in mammalian systems is quite scarce. It must be emphasised that bacteriophages are natural parasites of bacteria, which in turn are parasites or symbionts of mammals (including humans). Bacteriophages are constantly present in mammalian bodies and the environment in great amounts. On the other hand, the perspective of the possible use of bacteriophage preparations for antibacterial therapies in cancer patients generates a substantial need to investigate the effects of phages on cancer processes.

Results

In these studies the migration of human and mouse melanoma on fibronectin was inhibited by purified T4 and HAP1 bacteriophage preparations. The migration of human melanoma was also inhibited by the HAP1 phage preparation on matrigel. No response of either melanoma cell line to lipopolysaccharide was observed. Therefore the effect of the phage preparations cannot be attributed to lipopolysaccharide. No differences in the effects of T4 and HAP1 on melanoma migration were observed.

Conclusion

We believe that these observations are of importance for any further attempts to use bacteriophage preparations in antibacterial treatment. The risk of antibiotic-resistant hospital infections strongly affects cancer patients and these results suggest the possibility of beneficial phage treatment. We also believe that they will contribute to the general understanding of bacteriophage biology, as bacteriophages, extremely ubiquitous entities, are in permanent contact with human organisms.     

Plantaricins S and T, Two New Bacteriocins Produced by Lactobacillus plantarum LPCO10 Isolated from a Green Olive Fermentation

Twenty-six strains of Lactobacillus plantarum isolated from green olive fermentations were tested for cross-antagonistic activities in an agar drop diffusion test. Cell-free supernatants from four of these strains were shown to inhibit the growth of at least one of the L. plantarum indicator strains. L. plantarum LPCO10 provided the broadest spectrum of activity and was selected for further studies. The inhibitory compound from this strain was active against some gram-positive bacteria, including clostridia and propionibacteria as well as natural competitors of L. plantarum in olive fermentation brines. In contrast, no activity against gram-negative bacteria was detected. Inhibition due to the effect of organic acids, hydrogen peroxide, or bacteriophages was excluded. Since the inhibitory activity of the active supernatant was lost after treatment with various proteolytic enzymes, this substance could be classified as a bacteriocin, designated plantaricin S. Plantaricin S was also sensitive to glycolytic and lipolytic enzymes, suggesting that it was a glycolipoprotein. It exhibited a bactericidal and nonbacteriolytic mode of action against indicator cells. This bacteriocin was heat stable (60 min at 100 degrees C), active in a pH range of 3.0 to 7.0, and also stable in crude culture supernatants during storage. Ultrafiltration studies indicated that plantaricin S occurred as multimolecular aggregates and that the size of the smallest active form is between 3 and 10 kDa. In sodium dodecyl sulfate-polyacrylamide gels, plantaricin S migrated as a peptide of ca. 2.5 kDa. Maximum production of plantaricin S was obtained in a fermentor system in unregulated pH and log-phase cultures of L. plantarum LPCO10 in MRS broth plus 4% NaCl. In these culture conditions, a second bacteriocin (designated plantaricin T) was produced in late-stationary-phase cultures of L. plantarum LPCO10. On the basis of its biological activity, its sensitivity to various enzymes, and its molecular weight (lower than that of plantaricin S) as assessed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, plantaricin T appeared different from plantaricin S. Curing experiments with L. plantarum LPCO10 resulted in the appearance of variants that no longer produced either of the two bacteriocins but that were still immune to both of them.     

鼠李糖乳杆菌-格氏乳杆菌联用对阴道致病菌的抑制作用

目的本文通过提取细菌基因组进行16S rDNA PCR扩增和测序,分析菌株的进化树分支,鉴定一株乳酸菌菌株RD-0060并检测RD-0060与已有菌株RD-0046联用的抑菌能力和细胞粘附能力。方法结合现有菌株RD-0046(格氏乳杆菌,Lactobacillus gasseri),采用牛津杯法研究RD-0060单菌、RD-0060和RD-0046联用抑制致病菌的能力。通过共培养细菌和阴道上皮细胞VK2/E6E7,研究RD-0060单菌和RD-0060/RD-0046二联菌粘附能力。结果 RD-0060为鼠李糖乳杆菌(Lactobacillus rhamnosus),具有抑制阿托波菌、阴道加德纳菌和常见好氧型病菌的功能,对阴道上皮细胞也有较强的粘附能力;RD-0060和RD-0046二联菌的抑菌效果和细胞粘附能力比单菌株更强。结论鼠李糖乳杆菌和格氏乳杆菌联用能显著抑制阴道致病菌生长,并且能够大量粘附阴道细胞,而且两菌株联用有协同效果,具有良好的临床应用和开发前景。     

Isolation and partial characterization of bacteriocins produced by Lactobacillus gasseri JCM 2124

Four antibacterially active peptides (B1 to B4) were purified from the culture broth of L. gasseri JCM 2124. The B2 peptide (gassericin B2) was determined to be 4400 Da by mass spectrometry and partially sequenced. Gassericin B2 did not show any sequence similarities to other known bacteriocins. The B1 and B3 peptides shared identical sequences with two peptides of a two-component bacteriocin from Lactobacillus acidophilus. However, synergistic activity upon complementation of B1 and B3 was not observed. Based on amino acid sequencing and molecular mass, it is suggested that B1 and B4 peptides were derived from B3 (gassericin B3).     

Identification of Antioxidants Produced by Lactobacillus plantarum

We identified two compounds that demonstrated 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity from cultures of Lactobacillus plantarum. Spectroscopic analyses proved these compounds to be L-3-(4-hydroxyphenyl) lactic acid (HPLA) and L-indole-3-lactic acid (ILA). The respective EC₅₀ values for HPLA and ILA were 36.6 ± 4.3 mM and 13.4 ± 1.0 mM.     

Transformation of Folate-Consuming Lactobacillus gasseri into a Folate Producer

Five genes essential for folate biosynthesis in Lactococcus lactis were cloned on a broad-host-range lactococcal vector and were transferred to the folate auxotroph Lactobacillus gasseri. As a result L. gasseri changed from a folate consumer to a folate producer. This principle can be used to increase folate levels in many fermented food products.     

Identification and characterization of novel surface proteins in Lactobacillus johnsonii and Lactobacillus gasseri

We have identified and sequenced the genes encoding the aggregation-promoting factor (APF) protein from six different strains of Lactobacillus johnsonii and Lactobacillus gasseri. Both species harbor two apf genes, apf1 and apf2, which are in the same orientation and encode proteins of 257 to 326 amino acids. Multiple alignments of the deduced amino acid sequences of these apf genes demonstrate a very strong sequence conservation of all of the genes with the exception of their central regions. Northern blot analysis showed that both genes are transcribed, reaching their maximum expression during the exponential phase. Primer extension analysis revealed that apf1 and apf2 harbor a putative promoter sequence that is conserved in all of the genes. Western blot analysis of the LiCl cell extracts showed that APF proteins are located on the cell surface. Intact cells of L. johnsonii revealed the typical cell wall architecture of S-layer-carrying gram-positive eubacteria, which could be selectively removed with LiCl treatment. In addition, the amino acid composition, physical properties, and genetic organization were found to be quite similar to those of S-layer proteins. These results suggest that APF is a novel surface protein of the Lactobacillus acidophilus B-homology group which might belong to an S-layer-like family.     

Transformation of folate-consuming Lactobacillus gasseri into a folate producer

Five genes essential for folate biosynthesis in Lactococcus lactis were cloned on a broad-host-range lactococcal vector and were transferred to the folate auxotroph Lactobacillus gasseri. As a result L. gasseri changed from a folate consumer to a folate producer. This principle can be used to increase folate levels in many fermented food products.     

In Vitro Fermentation of Breast Milk Oligosaccharides by Bifidobacterium infantis and Lactobacillus gasseri

It has been proposed that human milk oligosaccharides (HMO) function as a prebiotic for bifidobacteria, yet this activity has not been adequately investigated. In this study, Bifidobacterium infantis was shown to ferment purified HMO as a sole carbon source, while another gut commensal, Lactobacillus gasseri, did not ferment HMO. Our results support the hypothesis that HMO selectively amplify bacterial populations in the infant intestine.     

Characterization of a Temperate Phage and Four Bacteriocins Produced by Nonpathogenic Clostridium Species

We previously reported the isolation of a temperate phage (named KT) and several bacteriocins (named clostocins) from strains of nonpathogenic Clostridium species. Later, the induction and some properties of the phage and four clostocins (A, B, C and D) were examined.

The phage was induced by UV light and mitomycin C. The phage had a polygonal head (about 85mμ in diameter) and a tail with contractile sheath (about 100mμ in length). Some other properties of the phage were also studied; plaque morphology, stability in salt solution, inactivation by UV light, pH stability, thermal inactivation, host-range and lysis of infected culture.

Clostocins A and D were partially induced by UV light and mitomycin C, whereas that of B and C were not. All clostocins failed to pass through a dialysis membrane, and were insensitive to UV light and to ribo- and deoxyribonuclease. They were destroyed by some proteolytic enzymes, but differences in degree of their susceptibility were observed among them. Clostocins A and D were very thermo-stable, whereas B and C were relatively thermo-labile. Clostocins A and D acted on some strains in the genus Clostridium, whereas B and C did on many strains in the family Bacillaceae.

There was no demonstrable serological relationship between phage KT and clostocin A, although they seemed to adsorb on the same bacterial receptor.