The linkage between the polysaccharide and mucopeptide components of the cell wall of Lactobacillus casei

1. The linkage between the polysaccharide and mucopeptide components of the cell wall of Lactobacillus casei is rapidly hydrolysed under mild acid-hydrolysis conditions. 2. The release of the polysaccharide is accompanied by the hydrolysis of an N-acetylhexosaminide linkage. The N-acetylhexosamine residue readily forms chromogen and it is concluded that it is substituted on C₍₃₎ by the adjacent sugar. 3. Continued heating of the polysaccharide in acid results in a slower release of reactive N-acetylhexosamine due to the hydrolysis of glycosidic linkages within the polysaccharide. 4. After the linkage between the polysaccharide and mucopeptide has been hydrolysed, acid phosphatase will release approx. 40% of the total phosphorus as inorganic phosphate. 5. It is concluded that the polysaccharide component of the cell wall is joined through its reducing end group to a phosphate grouping in the mucopeptide.     

Enhancement of host resistance against Listeria infection by Lactobacillus casei: efficacy of cell wall preparation of Lactobacillus casei

Cell wall, cytoplasm, polysaccharide, and peptidoglycan fractions prepared from Lactobacillus casei, L. plantarum, and L. acidophilus were examined for their efficacies to enhance resistance of host mice against Listeria monocytogenes infection. Intraperitoneal injections of those cellular fractions of L. casei led to elicitation of inflammatory cells in the peritoneal cavity and the efficacy was highest in the case of peptidoglycan. Macrophage ratio in the resultant peritoneal exudate cells was also highest in mice given peptidoglycan. Macrophages induced with cell wall fraction of L. casei showed the most potent phorbol myristate acetate (PMA)-triggered respiratory burst (chemiluminescence and O2- production determined on the basis of nitroblue tetrazolium reduction) followed by those elicited with peptidoglycan. All the macrophages induced with cell wall of L. casei (two strains) and L. acidophilus enhanced O2- production in response to PMA but L. plantarum did not enhance O2(-)-producing ability in such a manner. The L. casei-cell wall also enhanced in vitro listericidal activity of mouse peritoneal macrophages, but such an activity was not noted in the case of L. acidophilus-cell wall. When mice were intravenously given the cellular fractions 7 or 13 days before L. monocytogenes infection, cell wall fractions of L. casei caused the most potent protective activity. A weak protective activity was also found in peptidoglycan of L. casei. Therefore, the protective action of L. casei against L. monocytogenes infection in host mice may be attributed to cell wall compounds and partially to the peptidoglycan moiety.     

Structure of polysaccharide-peptidoglycan complex from the cell wall of Lactobacillus casei YIT9018

The isolation and analysis of the polysaccharide-peptidoglycan complexes of Lactobacillus casei YIT9018 are presented. Two polysaccharide-peptidoglycan complexes, PS-PG1 and PS-PG2, were solubilized from the heat-killed cell by treatment with N-acetylmuramidase. PS-PG1 was composed of glucose, rhamnose, and small amount of galactose and glucosamine. PS-PG2 was composed of glucose, rhamnose, galactosamine, and glucosamine. The ratio by weight of these fractions was about 1:8. PS-PG2 was analyzed in detail. Smith degradation and deamination of this complex yielded oligosaccharide units. The results of methylation analysis of these units and intact PS-PG2 led to the most probable structure of PS-PG2: (formula; see text)     

Guard cell wall: immunocytochemical detection of polysaccharide components

The composition of guard cell walls in sugar beet leaves (Beta vulgaris L.) was studied by using histochemical staining and immunocytochemical detection of cell wall antigens. The findings were compared with those in the walls of epidermal and mesophyll cells. Probing of leaf sections with monoclonal antibodies against pectins, terminal fucosyl residues linked alpha-(1-->2) to galactose, beta-(1-->3)-glucans and arabinogalactan-proteins revealed several specific features of guard cells. Pectic epitopes recognized by JIM7 were homogeneously distributed in the wall, whereas pectins recognized by JIM5 were not found in the walls themselves, but were abundant in the cuticular layer. Large amounts of molecules bearing terminal fucose were located predominantly in ventral and lateral guard cell walls. Much smaller amounts were detected in dorsal walls of these cells, as well as in the walls of pavement and mesophyll cells. Conspicuous accumulation of these compounds was observed in the vicinity of the guard cell plasmalemma, whereas labelling was scarce in the areas of the wall adjacent to the cell surface. The presence of callose clearly marked the ventral wall between the recently formed, very young guard cells. Callose also appeared in some mature walls, where it was seen as punctate deposits that probably reflected a specific physiological state of the guard cells. Large amounts of arabinogalactan-proteins were deposited within the cuticle, and smaller amounts of these proteoglycans were also detected in other tissues of the leaf. The histochemical and immunocytochemical structure of the guard cell wall is discussed in the light of its multiple functions, most of which involve changes in cell size and shape.     

Cell wall modifications during osmotic stress in Lactobacillus casei

AIMS: To study the modification of the cell wall of Lactobacillus casei ATCC 393 grown in high salt conditions. METHODS AND RESULTS: Differences in the overall structure of cell wall between growth in high salt (MRS + 1 mol l(-1) NaCl; N condition) and control (MRS; C condition) conditions were determined by transmission electronic microscopy and analytical procedures. Lactobacillus casei cells grown in N condition were significantly larger than cells grown under unstressed C condition. Increased sensitivity to mutanolysin and antibiotics with target in the cell wall was observed in N condition. Purified cell wall also showed the increased sensitivity to lysis by mutanolysin. Analysis of peptidoglycan (PG) from stressed cells showed that modification was at the structural level in accordance with a decreased PG cross-link involving penicillin-binding proteins (PBP). Nine PBP were first described in this species and these proteins were expressed in low percentages or presented a modified pattern of saturation with penicillin G (Pen G) during growth in high salt. Three of the essential PBP were fully saturated in N condition at lower Pen G concentrations than in C condition, suggesting differences in functionality in vivo. CONCLUSIONS: The results show that growth in high salt modified the structural properties of the cell wall. SIGNIFICANCE AND IMPACT OF STUDY: Advances in understanding the adaptation to high osmolarity, in particular those involving sensitivity to lysis of lactic acid bacteria.     

Properties of Lactose Plasmid pLY101 in Lactobacillus casei

A starter strain, Lactobacillus casei C257, was found to carry a lactose plasmid, pLY101. Restriction mapping showed that pLY101 DNA was 68.2 kilobases long. Since a non-lactose-utilizing variant of C257, MSK248, lost phospho-β-galactosidase (P-β-gal) activity and pLY101 DNA had a sequence(s) homologous to the streptococcal fragment including a P-β-gal gene, pLY101 is likely to encode a P-β-gal gene required for lactose metabolism in C257. MSK248 grew in galactose medium at a rate identical to that of C257 and retained phosphoenolpyruvate-dependent phosphotransferase system activity for lactose similar to that of C257. Therefore, the C257 chromosome appears to encode a complete set of genes for the lactose-phosphotransferase system and the predominant galactose metabolic pathway in C257. pLY101 DNA had a sequence homologous to a lactobacillus insertion sequence, ISL1, which mapped more than 12 kilobases from the sequence homologous to the streptococcal P-β-gal fragment.     

The isolation of oligosaccharides from the cell-wall polysaccharide of Lactobacillus casei, serological group C

1. A number of disaccharides and oligosaccharides have been isolated from the products of mild acid hydrolysis of the specific substance from Lactobacillus casei, serological group C. 2. The major disaccharide is O-β-d-glucopyranosyl-(1→3)-N-acetyl- d-galactosamine (B4) and evidence is presented for the structure of a tetrasaccharide composed of O-β-d-glucopyranosyl-(1→6)-d-galactose (B1) joined through its reducing end group to B4. 3. Disaccharide B1 is also a component of a trisaccharide O-β-d-glucopyranosyl-(1→6)-O-β- d-galactopyranosyl-(1→6)-N-acetyl-d-glucosamine (A7). 4. A number of other oligosaccharides have been shown to be related structurally. 5. The ability of certain of the oligosaccharides to inhibit the precipitin reaction has been studied. The disaccharide B1 is more effective as an inhibitor than gentiobiose and the trisaccharide A7 is considerably more effective than B1. 6. These results have been compared with those obtained previously for the composition of the cell wall.     

Cementing the wall: cell wall polysaccharide synthesising enzymes

The year under review has seen the first molecular characterisation, with proof of functionality, of Golgi membrane-bound glycosyltransferase enzymes catalysing the synthesis of non-cellulosic plant cell-wall polysaccharides.     

Citrate metabolism in Lactobacillus casei and Lactobacillus plantarum

Information on the factors influencing citrate metabolism in lactobacilli is limited and could be useful in understanding the growth of lactobacilli in ripening cheese. Citrate was not used as an energy source by either Lactobacillus casei ATCC 393 or Lact. plantarum 1919 and did not affect the growth rate when co-metabolized with glucose or galactose. In growing cells, metabolism of citrate was minimal at pH 6 but significant at pH 4·5 and was greater in cells co-metabolizing galactose than in those co-metabolizing glucose or lactose. In non-growing cells, optimum utilization of citrate also occurred at pH 4·5 and was not increased substantially by the presence of fermentable sugars. In both growing and non-growing cells, acetate and acetoin were the major products of citrate metabolism; pyruvate was also produced by non-growing cells and was transformed to acetoin once the citrate was exhausted. Citrate was metabolized more rapidly than sugar by non-growing cells; the reverse was true of growing cells. Citrate metabolism by Lact. plantarum 1919 and Lact. casei ATCC 393 increased six- and 22-fold, respectively, when the cells were pre-grown on galactose plus citrate than when pre-grown on galactose only. This was probably due to induction of citrate lyase by growth on citrate plus sugar. These results imply that lactobacilli, if present in large enough numbers, can metabolize citrate in ripening cheese in the absence of an energy source.     

Lectin microarray reveals binding profiles of Lactobacillus casei strains in a comprehensive analysis of bacterial cell wall polysaccharides

We previously showed a pivotal role of the polysaccharide (PS) moiety in the cell wall of the Lactobacillus casei strain Shirota (YIT 9029) as a possible immune modulator (E. Yasuda M. Serata, and T. Sako, Appl. Environ. Microbiol. 74:4746-4755, 2008). To distinguish PS structures on the bacterial cell surface of individual strains in relation to their activities, it would be useful to have a rapid and high-throughput methodology. Recently, a new technique called lectin microarray was developed for rapid profiling of glycosylation in eukaryotic polymers and cell surfaces. Here, we report on the development of a simple and sensitive method based on this technology for direct analysis of intact bacterial cell surface glycomes. The method involves labeling bacterial cells with SYTOX Orange before incubation with the lectin microarray. After washing, bound cells are directly detected using an evanescent-field fluorescence scanner in a liquid phase. Using this method, we compared the cell surface glycomes from 16 different strains of L. casei. The patterns of lectin-binding affinity of most strains were found to be unique. There appears to be two types of lectin-binding profiles: the first is characterized by a few lectins, and the other is characterized by multiple lectins with different specificities. We also showed a dramatic change in the lectin-binding profile of a YIT 9029 derivative with a mutation in the cps1C gene, encoding a putative glycosyltransferase. In conclusion, the developed technique provided a novel strategy for rapid profiling and, more importantly, differentiating numerous bacterial strains with relevance to the biological functions of PS.     

Protoplast fusion between Lactobacillus casei and Lactobacillus acidophilus

Summary From the fusion between Lactobacillus casei and Lactobacillus acidophilus, 8 fusants were selected: Four were able to ferment maltose, lactose, galactose and mannose, but two had greater abilities of acid production than parents. Increased values of up to 7.6–8 % in -galactosidase activity were obtained from two when compared to that of L. acidophilus, whereas another 2 had activities of 800 and 548 nmol/mg protein/min comparable to that of L casei giving a value of 400 nmol/mg protein/min in phospho--galactosidase activity.     

Release of aminopeptidase from Lactobacillus casei sp. casei by cell disruption in a Microfluidizer

Cell disruption in a Microfluidizer was a function of both operating pressure and number of passes. The operating pressure had greater effect on the disruption than did the number of passes as indicated by the magnitude of the constants from the cell disruption equation. Protein release correlated with aminopeptidase release by Lactobacillus casei sp. casei. The optimum operating pressure for enzyme extraction was 76 MPa with loss of enzyme activity about 15 to 20%.