Presence of Active and Inactive Molecules of a Cell Wall-Associated Proteinase in Lactobacillus helveticus CP790

Monoclonal antibodies against a cell wall-associated 45-kDa proteinase from Lactobacillus helveticus CP790 were prepared and used for an immunoblotting analysis of the cell wall extract of CP790. They were found to react with an unidentified 46-kDa protein as well as the 45-kDa proteinase. The 46-kDa protein was copurified with the 45-kDa proteinase by affinity column chromatography using antibody-fixed Sepharose and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then extracted from the gels. An elution profile of the cyanogen bromide digest of the purified 46-kDa protein obtained by reversed-phase high-performance liquid chromatography was identical to that of the 45-kDa proteinase except for one peak. An analysis of the N-terminal 21-amino-acid sequence revealed that the 46-kDa protein possesses an extra 7 amino acids at the N terminus of the 45-kDa proteinase. The 46-kDa protein was produced at constant levels during fermentation in a skim milk medium, while the 45-kDa protein was mainly observed in the middle of the exponential phase of growth and was produced in proportion to the proteinase activity. Moreover, only the 46-kDa protein was detected in the crude extract of L. helveticus CP791, a variant strain of CP790 defective in proteinase activity. These data strongly suggest that the 46-kDa protein is a precursor, inactive form of the 45-kDa proteinase.     

DNA restriction endonuclease cleavage patterns,DNA sequence similarity and phenotypical characteristics in some strains of Lactobacillus helveticus and Lactobacillus jugurti

Physiological characteristics, deoxyribonucleic acid (DNA) base composition (% guanine + cytosine; % GC), DNA sequence similarity (% DNA-DNA hybridization) and DNA restriction endonuclease cleavage patterns of two strains of Lactobacillus helveticus and four strains of Lactobacillus jugurti were examined.All the strains investigated were closely related genetically, having DNA-DNA hybridization values ranging from 89–100%. Nevertheless, these strains can be differentiated from one another on the basis of the digestion of their DNA by specific restriction endonucleases, such as Bam HI, Eco RI and Hind III. The DNA of these strains shows clear, reproducible and distinct cleavage patterns. Cleavage patterns of DNA from strains L. jugurti S.35.19 and S.36.2 were found to be similar. These findings suggest that fingerprinting of DNA by restriction endonuclease cleavage might provide, in addition to the conventional methods, a useful tool for the characterization of closely related microorganisms at the strain level.     

Patterns of Reactivity between a Panel of Monoclonal Antibodies and Forage Rhizobium Strains

A panel of 11 monoclonal antibodies raised against vegetative cells of Rhizobium leguminosarum biovar trifolii or Rhizobium meliloti was tested by enzyme-linked immunosorbent assay for reactivity with 47 strains of R. leguminosarum biovar trifolii and 60 strains of R. meliloti. The goal of the study was to define the degree of specificity associated with each antibody and to gain an understanding of the amount of antigenic diversity found among the strains and between the species. Each antibody was tested against each Rhizobium strain in four forms: washed steamed cells, washed unsteamed cells, cell-free culture broth, and nodule squash material. Each antibody showed a different pattern of reactivity among the 107 strains. One of each of the antibodies developed against R. meliloti and R. leguminosarum biovar trifolii reacted in a highly specific manner with cells or antigen from the immunogenic strain only. Nine of the antibodies recognized secreted as well as cellular antigen from many of the strains. Analysis of patterns of reactivity between the 107 strains and the 11 antibodies separated the strains into 28 groups of which 12 were represented by one strain only.     

Molecular cloning and sequence analysis of a gene encoding an extracellular proteinase from Lactobacillus helveticus CP790

A 2.6-kilobase HaeIII DNA fragment corresponding to an extracellular proteinase gene (prtY) was cloned from chromosomal DNA of Lactobacillus helveticus CP790 in Escherichia coli using a pKK223-3 vector. The transformant expressed a 48-kDa protein that reacts with monoclonal antibodies specific to the proteinase and seemed to be a pre-proproteinase, but had no proteolytic activity. About 1.6 kilobases of the 2.6-kilobase DNA fragment, which contained the complete gene for the proteinase was sequenced. Sequence analysis found an open reading frame with a capacity to encode a protein of 449 amino acids. The coding region contained a Gram-positive-type signal peptide of 30 amino acids. The N-terminal sequences of the proproteinase and the mature proteinase have been observed in the polypeptide at position + 31 and + 38. The putative amino acid sequence showed a significant similarity to a surface layer protein of L. helveticus and Lactobacillus acidophilus in the amino terminal signal sequence and carboxyl terminus.     

Effect of medium composition on the ultrastructure of Lactobacillus strains

The growth of some locally isolated Lactobacillus strains forming D(-) or L(+) lactic acid, Lactobacillus helveticus ATCC 15009 and Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 was examined in different media. L. helveticus and Lactobacillus LBL strains formed atypical protoplast-like cells in LAPT medium, sensitive to SDS and proteinase. Specific morphological changes in the cell wall structure of these variants were revealed by transmission and scanning electron microscopy. The effect of glucose and various salts on their appearance was investigated. The prevalent role of metal cations, especially of Mg²⁺, was established.     

Monoclonal Antibodies to the Cell-Wall-Associated Proteinase of Lactococcus lactis subsp. cremoris Wg2

Twelve monoclonal antibodies directed to the cell-wall-associated proteinase of Lactococcus lactis subsp. cremoris Wg2 were isolated after immunization of BALB/c mice with a partially purified preparation of the proteinase. The monoclonal antibodies reacted with the 126-kilodalton proteinase band in a Western immunoblot. All but one of the monoclonal antibodies reacted with protein bands with a molecular weight below 126,000, possibly degradation products of the proteinase. The monoclonal antibodies could be divided into six groups according to their different reactions with the proteinase degradation products in the Western blot. Different groups of monoclonal antibodies reacted with different components of the L. lactis subsp. cremoris Wg2 proteinase. Crossed immunoelectrophoresis showed that monoclonal antibody groups I, II, and III react with proteinase component A and that groups IV, V, and VI react with proteinase component B. The isolated monoclonal antibodies cross-reacted with the proteinases of other L. lactis subspecies. Monoclonal antibodies of group IV cross-reacted with proteinase component C of other L. lactis subsp. cremoris strains. The molecular weight of the proteinase attached to the cells of L. lactis subsp. cremoris Wg2 was 200,000, which is different from the previously reported values. This could be analyzed by immunodetection of the proteinase on a Western blot. This value corresponds to the molecular weight calculated from the amino acid sequence of the cloned L. lactis subsp. cremoris Wg2 proteinase gene.     

Functionality of the S-layer protein from the probiotic strain <Emphasis Type="Italic">Lactobacillus helveticus</Emphasis> M92

The objective of this study was the characterisation of the S-layer protein (SlpA) and its functional role in the probiotic activity of Lactobacillus helveticus M92. SlpA was isolated and identified by SDS-PAGE LC-MS/MS analysis. The slpA gene encoding the SlpA from L. helveticus M92 was sequenced and compared with other well characterised slpA genes. Sequence similarity searches revealed high homology with the SlpA of Lactobacillus strains. Purified SlpA showed significantly better immunomodulatory effects in orally immunised mice than L. helveticus M92 cells after SlpA removal. SlpA is involved in the autoaggregation of L. helveticus M92 cells and coaggregation of L. helveticus M92 with S. Typhimurium FP1 as these processes were negatively affected after SlpA removal from the cell surface. Therefore, the influence of oral treatment with L. helveticus M92 on an oral infection of mice by S. Typhimurium FP1 was investigated. Following the oral immunization of mice, with viable L. helveticus M92 and S. Typhimurium FP1 cells, the concentration in the luminal contents of total S-IgA and specific anti-Salmonella S-IgA antibodies, from all immunized mice was significantly higher compared to the control group or a group of mice infected only with S. Typhimurium FP1. These results demonstrate that the observed reduced infection by S. Typhimurium FP1 in mice with L. helveticus M92 is associated with competitive exclusion in the intestinal tract and enhanced immune protection conferred by the L. helveticus M92 and its SlpA.     

Modulation of the Cell-Surface Proteinase Activity of Thermophilic Lactobacilli by the Peptide Supply

The proteolytic system of thermophilic lactobacilli is considered important for bacterial nutrition as well as for the formation of flavor and texture in fermented products. We investigated the influence of peptide content on the cell surface proteinase and intracellular aminopeptidase activities from seven thermophilic lactobacilli strains. The proteinase activities were remarkably reduced in cells grown in the peptide-rich medium MRS or in a chemically defined medium supplemented with Casitone compared with those found in a synthetic medium. The degree of inhibition observed was strain dependent. When proteinase activities were analyzed by their hydrolytic patterns of α- and β-casein degradation, four types of P_III-caseinolytic cleavage specificity were distinguished. Lactobacillus helveticus strains possessed aminopeptidase activities with broader specificity than those found in L. delbrueckii subsp. lactis strains. However, the aminopeptidase activities were not influenced by the peptide content of the medium. Received: 1 February 2002 / Accepted: 27 February 2002     

A Lactobacillus helveticus-Specific DNA Probe Detects Restriction Fragment Length Polymorphisms in This Species

A cloned 2-kb EcoRI fragment (fragment f) from a 34-kb plasmid of Lactobacillus helveticus CNRZ 1094 was shown by dot blot to specifically hybridize to total DNAs of 75 L. helveticus strains. No hybridization was found with L. acidophilus, L. crispatus, L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, L. gasseri, or L. intestinalis strains. When Southern blots of EcoRI digests of L. helveticus strains were probed with fragment f, these strains displayed restriction fragment length polymorphisms on the basis of which they could be grouped into several clusters.     

Doxycycline-Dependent Inducible and Reversible RNA Interference Mediated by a Single Lentivirus Vector

Most fermented milk prepared by strains of Lactobacillus helveticus showed significant antihypertensive effect in spontaneously hypertensive rats (SHR) by oral administration. However, milk fermented by other species of lactic acid bacteria did not show significant antihypertensive effects. Most of the whey fractions of the milk fermented by L. helveticus or Lactobacillus delbrueckii subsp. bulgaricus showed higher angiotensin I-converting enzyme (ACE) inhibitory activity than the activity of milk fermented by other species. Proteolytic activity in cell wall and peptide content of the fermented milk were higher in L. helveticus strains than other species.     

Molecular Identification of Potentially Probiotic Lactobacilli

The rRNA-targeted oligonucleotide probes are useful for the identification of Lactobacillus acidophilus, L. gasseri, L. johnsonii, L. crispatus, and L. amylovorus. However, the oligonucleotide probe designed for L. helveticus hybridized with nucleic acids of type strains of L. gallinarum and L. helveticus. Hence, the similarity among the 73 strains of lactobacilli was evaluated on the basis of their randomly amplified polymorphic DNA (RAPD) profiles derived from five single-primer reactions. These strains were grouped into seven clusters at a similarity level of 30%, which corresponded to six separate species of the L. acidophilus complex (L. johnsonii, L. gallinarum, L. amylovorus, L. crispatus, L. acidophilus, and L. gasseri, respectively) and L. helveticus. For the first time, strains of L. gallinarum were characterized by RAPD and PFGE analyses. The genome length in that species was estimated to be near 1.45 Mb with the summation of ApaI fragments, and near 1.95 Mb with the summation of SmaI fragments. Received: 1 July 1999 / Accepted: 2 August 1999     

Use of SDS-PAGE of cell-wall proteins for rapid differentiation of Lactobacillus delbrueckii subsp. lactis and Lactobacillus helveticus

Cell-wall protein profiles of different strains of Lactobacillus helveticus and L. delbrueckii subsp. lactis isolated from regional cheeses were studied by SDS-PAGE. The patterns were highly reproducible and the presence of numerous bands with molecular weight ranging from 14 to 160 kDa allowed L. delbrueckii subsp. lactis to be differentiated from L. helveticus. The method is a reliable and rapid way to identify thermophilic lactobacilli.     

Purification and Properties of Extracellular Carboxyl Proteases of Acid-tolerant Bacteria,Isolated from Compost

Four strains of acid-tolerant and protein-using bacteria were isolated from compost. Two of them, Gram-negative strains MB8 and MB11, were identified as a new genus close to Stenotrophomonas species MB8 and Burkholderia species MB11, respectively. Both bacteria produced extracellular carboxyl proteases (CP) in acid-casein-starch medium. The enzymes, termed CP MB8 and CP MB11, purified through ion exchange and gel filtration chromatographies had molecular weights of 61,000 (CP MB8) and 36,000 (CP MB11) as estimated by SDS-PAGE, and showed optimum activities with hemoglobin as a substrate at pH 3.5 (CP MB8) and pH 3.7 (CP MB11) at 55°C. Both of the enzymes were not inhibited by pepstatin, DAN, or EPNP. These results suggest that both enzymes are members of the pepstatin-insensitive carboxyl proteinase family (EC 3.4.23.33), except for a larger molecular weight of the CP MB8 enzyme.     

Diversity in proteinase specificity of thermophilic lactobacilli as revealed by hydrolysis of dairy and vegetable proteins

Ability of industrially relevant species of thermophilic lactobacilli strains to hydrolyze proteins from animal (caseins and β-lactoglobulin) and vegetable (soybean and wheat) sources, as well as influence of peptide content of growth medium on cell envelope-associated proteinase (CEP) activity, was evaluated. Lactobacillus delbrueckii subsp. lactis (CRL 581 and 654), L. delbrueckii subsp. bulgaricus (CRL 454 and 656), Lactobacillus acidophilus (CRL 636 and 1063), and Lactobacillus helveticus (CRL 1062 and 1177) were grown in a chemically defined medium supplemented or not with 1 % Casitone. All strains hydrolyzed mainly β-casein, while degradation of α_s-caseins was strain dependent. Contrariwise, κ-Casein was poorly degraded by the studied lactobacilli. β-Lactoglobulin was mainly hydrolyzed by CRL 656, CRL 636, and CRL 1062 strains. The L. delbrueckii subsp. lactis strains, L. delbrueckii subsp. bulgaricus CRL 656, and L. helveticus CRL 1177 degraded gliadins in high extent, while the L. acidophilus and L. helveticus strains highly hydrolyzed soy proteins. Proteinase production was inhibited by Casitone, the most affected being the L. delbrueckii subsp. lactis species. This study highlights the importance of proteolytic diversity of lactobacilli for rational strain selection when formulating hydrolyzed dairy or vegetable food products.     

Productivity of hydrolytic enzymes by mycorrhizal mushrooms

To survey the potential for production of extracellular hydrolytic enzymes by mycorrhizal mushrooms, productivities of these exo-enzymes from mycelia on potato-dextrose liquid medium were determined.Tricholoma matsutake produced relatively high levels of CM-cellulase and avicelase activities in all test strains. It also produced higher activity of acid proteinase than neutral proteinase. Its xylanase activities seemed to be higher than those of the other carbohydrases. The productivities ofLyophyllum shimeji strains were at similar levels to those ofT. matsutake strains. CM-cellulase and avicelase activities ofL. shimeji were higher than those ofT. matsutake. Neutral proteinase inL. shimeji strains showed higher activity levels than acid proteinase. The relative productivities of hydrolytic enzymes between the groups of mycorrhizal mushrooms and wood-rotting mushrooms were also examined.T. matsutake andL. shimeji both produce many kinds of hydrolytic enzymes in their culture broth, and the potential for production of hydrolytic enzymes by mycorrhizal mushrooms was judged to be relatively high.     

Plasmodium vivax:A Monoclonal Antibody Recognizes a Circumsporozoite Protein Precursor on the Sporozoite Surface

Gonzalez-Ceron, L., Rodriguez, M. H., Wirtz, R. A., Sina, B. J., Palomeque, O. L., Nettel, J. A., and Tsutsumi, V. 1998.Plasmodium vivax:A monoclonal antibody recognizes a circumsporozoite protein precursor on the sporozoite surface.Experimental Parasitology90, 203–211. The major surface circumsporozoite (CS) proteins are known to play a role in malaria sporozoite development and invasion of invertebrate and vertebrate host cells.Plasmodium vivaxCS protein processing during mosquito midgut oocyst and salivary gland sporozoite development was studied using monoclonal antibodies which recognize different CS protein epitopes. Monoclonal antibodies which react with the CS amino acid repeat sequences by ELISA recognized a 50-kDa precursor protein in immature oocyst and additional 47- and 42-kDa proteins in older oocysts. A 42-kDa CS protein was detected after initial sporozoite invasion of mosquito salivary glands and an additional 50-kDa precursor CS protein observed later in infected salivary glands. These data confirm previous results with otherPlasmodiumspecies, in which more CS protein precursors were detected in oocysts than in salivary gland sporozoites. A monoclonal antibody (PvPCS) was characterized which reacts with an epitope found only in the 50-kDa precursor CS protein. PvPCS reacted with allP. vivaxsporozoite strains tested by indirect immunofluorescent assay, homogeneously staining the sporozoite periphery with much lower intensity than that produced by anti-CS repeat antibodies. Immunoelectron microscopy using PvPCS showed that the CS protein precursor was associated with peripheral cytoplasmic vacuoles and membranes of sporoblast and budding sporozoites in development oocysts. In salivary gland sporozoites, the CS protein precursor was primarily associated with micronemes and sporozoite membranes. Our results suggest that the 50-kDa CS protein precursor is synthesized intracellularly and secreted on the membrane surface, where it is proteolytically processed to form the 42-kDa mature CS protein. These data indicate that differences in CS protein processing in oocyst and salivary gland sporozoites development may occur.     

Genetic structure and phylogenetic relationships of Ralstonia solanacearum strains from diverse origins in Guangdong Province,China

Bacterial wilt, caused by Ralstonia solanacearum species complex is a key yield‐limiting factor on crops in Guangdong province, China. The genetic diversity of 110 R. solanacearum strains collected from 16 host plants in different areas of Guangdong province was analysed using biovar and phylotype classification schemes. Of 110 strains, fifty‐five strains belong to biovar 3, fifty‐two strains belong to biovar 4, two strains belong to biovar 2 and one strain belonged to biovar 1. Phylotype‐specific multiplex PCR showed that 108 strains belonged to phylotype I (biovars 1, 3, 4) and two strains belonged to phylotype II (biovar 2). The result of phylogenetic relationships analysis based on egl gene sequences demonstrated that 108 strains of phylotype I were grouped into nine previously described sequevars and a new sequevar 57, and two strains of phylotype II were grouped into sequevar 1. Sequevars 15, 34 and 44 widely distributed in Guangdong were predominant sequevars. Sequevar 45 was first reported on potato and pumpkin in China. These results revealed the genetic structure and phylogenetic relationships of R. solanacearum population in Guangdong and will be helpful in bacterial wilt‐resistance breeding.     

Chromatographic Study of Spirosin by Use of Monoclonal Antibodies to Spirosin of Yersinia enterocolitica

Two monoclonal antibodies (MAbs) reacting with spirosins from Enterobacteriaceae were obtained in a course of screening MAbs to spirosin from Yersinia enterocolitica SYT-11-72 (YE72). The antibodies were designated MAbs-S44 and S50. They were IgG_2b and IgG_2a, respectively, both with k light chains. On Western blotting after limited proteolysis of YE72 spirosin with Staphylococcus aureus V8 protease, they reacted markedly with peptide fragments of 27 and 35 kDa, suggesting the presence of an antigenic determinant on the fragments. When supernatant cell lysate from Escherichia coli K12 was chromatographed on DEAE-cellulose and Sepharose CL-6B columns successively, a 96-kDa protein with alcohol dehydrogenase (ADH) activity was always associated with reactivity to MAb-S50. These findings combined with N-terminal amino acid sequences clearly indicate the identity of spirosin to ADH in E. coli.     

Molecular Analysis of the Gal/GalNAc Adhesin of Entamoeba histolytica1

Attachment of Entamoeba histolytica to colonic epithelium and a variety of other target cells is mediated by a galactosc/N-acetyl D-galactosamine (Gal/GalNAc) inhibitable adhesin. Seven monoclonal antibodies specific for nonoverlapping epitopes of the 170 kDa subunit have been shown to have distinct effects on adherence. Four of these monoclonal antibodies inhibit or have no effect on amebic adherence while two others enhance amebic adherence. The epitopes recognized by these seven monoclonal antibodies have been mapped to the extracellular cysteine rich region of the 170 kDa subunit. The conformational nature of the epitopes was examined by testing monoclonal antibody reactivity with isolated regions of the 170 kDa subunit expressed as fusion proteins in E. coli and also with denatured native adhesin. These analyses suggested that three of monoclonal antibodies recognized conformational epitopes while the remaining four recognized linear epitopes. The mapping of these monoclonal antibodies have identified functionally important regions of the Gal/GalNAc adhesin and have also shown that recombinant Gal/GalNAc adhesin, when expressed in E. coli, retained at least some of its native conformation.     

Antibiotic resistance of potential probiotic bacteria of the genus <Emphasis Type="Italic">Lactobacillus</Emphasis> from human gastrointestinal microbiome

Thirteen Lactobacillus strains isolated from the gastrointestinal microbiome of people from the territory of the former Soviet Union have been studied for resistance to 15 antibiotics of different nature, namely, penicillins, aminoglycosides, macrolides, lincosamides, tetracyclines, chloramphenicol, and rifampicin. The strains included four strains of L. plantarum, four of L. helveticus, three of L. casei/paracasei, one of L. rhamnosus, and one of L. fermentum. All strains showed relative sensitivity to ampicillin, chloramphenicol, rifampicin, roxithromycin, erythromycin, and azithromycin, while none of them were sensitive to all tested antibiotics. L. plantarum strains had the broadest resistance spectra: one strain was resistant to tetracycline and three aminoglycosides and three strains were resistant to tetracycline and five aminoglycosides; one strain demonstrated high resistance to clindamycin and two strains to lincomycin. At the same time, two L. plantarum strains demonstrated resistance to benzylpenicillin coupled with sensitivity to ampicillin, another β-lactam antibiotic. Such resistance was clearly not related to the β-lactamase activity and could be explained by a specific mutation in one of the penicillin-binding proteins of the cell wall. Strains of L. helveticus, L. casei/paracasei, L. rhamnosus, and L. fermentum exhibited cross resistance to two to five different aminoglycosides. A PCR test of the resistance determinants for the widely clinically used antibiotics, tetracycline, chloramphenicol, and erythromycin revealed the presence of the tetM gene of conjugative transposon in L. casei/paracasei and two L. helveticus strains. Nucleotide sequence analysis of the amplified tetM fragments demonstrated their high homology with the tetM genes of Enterococcus faecalis and Streptococcus pneumoniae. The strains carrying tetM were tested for the genes of replication and conjugative transfer of plasmids in lactic acid bacteria. The results indicated that these strains contain genes identical or highly homologous to the rep and trsK genes of the plca36 plasmid and rep gene of the pLH1 and pLJ1 plasmids of lactic acid bacteria. The tetM gene is probably not expressed in strains sensitive to the corresponding antibiotic. However, the investigated lactobacilli cannot be directly used as probiotics, as they may serve as a source of genes for antibiotic resistance in the human microbiome.