Reassembly of a regularly arranged protein in the cell wall of Lactobacillus buchneri and its reattachment to cell walls: chemical modification studies

Regularly arranged protein (RA protein) isolated from the cell wall of Lactobacillus buchneri was chemically modified by amidination, acetylation, succinylation, and amidation. The modified RA proteins were examined for their ability to reassemble into a regular array and to reattach to the cell walls from which the regular array had been detached. Only amidinated RA protein could be either reassembled into a regular array or reattached to the cell walls; RA proteins modified by the other methods lost the ability for both reassembly and reattachment. The unmodified RA protein could be reattached to periodate-oxidized cell walls, but not to methylated ones. These results suggest that the positive charge of the amino group as well as the negative charge of the carboxyl group of RA protein plays an important role(s) in morphogenesis of the hexagonal array and in its attachment to the underlying cell wall layer. The periodate-insensitive lone hydroxyl groups of the neutral polysaccharide molecule in the cell wall seem to be the receptor sites for RA protein in the attachment to the cell wall.     

Demonstration and preliminary characterization of a regular array in the cell wall of Clostridium difficile

Abstract The presence of a regular array (RA) was demonstrated on the outer layer of the cell wall in Clostridium difficile GAI0714 by electron microscopy. The RA was composed of squarely arranged subunits with a center-to-center spacing of about 8.2 nm. The outer wall layer carrying the RA was isolated from the wall fragments of early log-phase cells by autolysis. The outer wall layer was composed of two main proteins with apparent M _rs of about 45 000 and 32 000 upon sodiumdodecylsul-fate-polyacrylamide gel electrophoresis (SDS-PAGE). Similar RAs were also present in the cell walls of the other 9 strains of C. difficile . These strains were divided into two groups on the basis of the wall protein composition: one containing M _r 45 000–47 000 and 32 000 proteins and the other containing M _r 42 000 and 38 000 proteins.     

Insights into the completely annotated genome of Lactobacillus buchneri CD034, a strain isolated from stable grass silage

Lactobacillus buchneri belongs to the group of heterofermentative lactic acid bacteria and is a common member of the silage microbiome. Here we report the completely annotated genomic sequence of L. buchneri CD034, a strain isolated from stable grass silage. The whole genome of L. buchneri CD034 was sequenced on the Roche Genome Sequencer FLX platform. It was found to consist of four replicons, a circular chromosome, and three plasmids. The circular chromosome was predicted to encode 2319 proteins and contains a genomic island and two prophages which significantly differ in G+C-content from the remaining chromosome. It possesses all genes for enzymes of a complete phosphoketolase pathway, whereas two enzymes necessary for glycolysis are lacking. This confirms the classification of L. buchneri CD034 as an obligate heterofermentative lactic acid bacterium. A set of genes considered to be involved in the lactate degradation pathway and genes putatively involved in the breakdown of plant cell wall polymers were identified. Moreover, several genes encoding putative S-layer proteins and two CRISPR systems, belonging to the subclasses I-E and II-A, are located on the chromosome. The largest plasmid pCD034-3 was predicted to encode 57 genes, including a putative polysaccharide synthesis gene cluster, whereas the functions of the two smaller plasmids, pCD034-1 and pCD034-2, remain cryptic. Phylogenetic analysis based on sequence comparison of the conserved marker gene rpoA reveals that L. buchneri CD034 is more closely related to Lactobacillus hilgardii strains than to Lactobacillus brevis and Lactobacillus plantarum strains. Comparison of the L. buchneri CD034 core genome to other fully sequenced and closely related members of the genus Lactobacillus disclosed a high degree of conservation between L. buchneri CD034 and the recently sequenced L. buchneri strain NRRL B-30929 and a more distant relationship to L. buchneri ATCC 11577 and L. brevis ssp. gravesensis ATCC 27305, which cluster together with L. hilgardii type strain ATCC 8290. L. buchneri CD034 genome information will certainly provide the basis for further postgenome studies with the objective to optimize application of the strain in silage production.     

Retinoic Acid and Its Geometrical Isomers Block Both Growth and Fusion of L6 Myoblasts by Modulating the Expression of Protein Kinase A

All-trans retinoic acid (RA) and its geometrical isomers, such as 9-cis RA, 13-cis RA, and 9,13-di-cis RA, strongly inhibited both growth and fusion of L6 myoblasts. However, illumination of white light diminished their inhibitory activity on membrane fusion with little effect on cell growth. During myogenic differentiation, the intracellular level of cAMP decreased whereas the total activity of protein kinase A as well as the protein level of its regulatory subunit I (RI) and catalytic subunit (C) increased. RAs raised the intracellular level of cAMP by over 3-fold, but decreased the total activity of protein kinase A. Like RAs, dibutyryl-cAMP inhibited myoblast fusion and reduced the expression of both RI and C subunits. These results suggest that RAs negatively modulate the differentiation of L6 myoblasts by increasing the intracellular level of cAMP, which may in turn down-regulate the expression of protein kinase A and hence its activity.     

Comparative characterization of spirosomes isolated from Lactobacillus brevis, Lactobacillus fermentum, and Lactobacillus buchneri

Spirosomes, cytoplasmic fine spirals, were isolated and purified from Lactobacillus brevis ATCC 8287, L. fermentum F-1, and L. buchneri ATCC 4005, and their morphological, biochemical, and immunological properties were investigated. The spirosomes of these lactobacilli were morphologically indistinguishable from one another, and they had the same buoyant density of 1.320 g/cm3 in CsCl. All of the spirosomes were composed of a single protein, spirosin, with an apparent molecular weight of about 95,000 for L. brevis and L. fermentum and of about 96,000 for L. buchneri as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The spirosins from the three lactobacilli were compared by peptide mapping on SDS-PAGE after cleavage with N-chlorosuccinimide and limited proteolysis with Staphylococcus aureus V8 protease. The peptide map of the L. brevis spirosin was identical with that of the L. fermentum spirosin, whereas it was markedly different from the L. buchneri spirosin. The amino acid composition of the L. brevis spirosin was almost similar to that of the L. fermentum spirosin, while it differed appreciably from the L. buchneri spirosin. Using antiserum against the L. brevis spirosin, immunodiffusion test revealed that the antigenicity of the spirosomes from L. brevis was identical with that from L. fermentum, whereas it was partially different from that from L. buchneri.     

Digestion and Gastrointestinal Absorption of the 14–16-kDa Rice Allergens

The digestibility and gastrointestinal absorption of 14–16-kDa rice allergens (RAs) were investigated. RAs and bovine serum albumin (BSA) were first evaluated for their digestibility. BSA was digested completely by in vitro incubation with some proteases, but RAs remained almost intact. Administered orally (20 mg per mouse), intact RAs were clearly detected in the small intestine even 60 min after the administration, the amount of total RAs in the small intestine being estimated to be 0.59 mg. RAs were then biotinylated and infused into the duodenal lumen of anesthetized mice, and portal blood was collected. The RA concentrations in the portal plasma were respectively estimated to be 0.4–0.9 and 0.3–2.5 μg/ml for 0.4 and 4 mg doses. These results suggest that RAs are highly resistant to digestive enzymes and that about 1/100 of orally administered RAs remain intact in the small intestine, while at least 1/1,000–1/10,000 is absorbed and delivered into circulated blood.     

Digestion and gastrointestinal absorption of the 14-16-kDa rice allergens

The digestibility and gastrointestinal absorption of 14-16-kDa rice allergens (RAs) were investigated. RAs and bovine serum albumin (BSA) were first evaluated for their digestibility. BSA was digested completely by in vitro incubation with some proteases, but RAs remained almost intact. Administered orally (20 mg per mouse), intact RAs were clearly detected in the small intestine even 60 min after the administration, the amount of total RAs in the small intestine being estimated to be 0.59 mg. RAs were then biotinylated and infused into the duodenal lumen of anesthetized mice, and portal blood was collected. The RA concentrations in the portal plasma were respectively estimated to be 0.4-0.9 and 0.3-2.5 microg/ml for 0.4 and 4 mg doses. These results suggest that RAs are highly resistant to digestive enzymes and that about 1/100 of orally administered RAs remain intact in the small intestine, while at least 1/1,000-1/10,000 is absorbed and delivered into circulated blood.     

Sensitivity of some Lactobacillus species of the subgenus Betabacterium to the action of bacteriocins from different species of Lactobacillus]

Sensitivity of 84 cultures of L. fermenti, 43 cultures of L. brevis, 13 cultures of L. buchneri and 2 cultures of L. cellobiosus to the effect of 39 types of bacteriocins produced by various species of lactobacilli was studied with the method of delayed antagonism. All the cultures of L. brevis, L. buchneri and L. cellobiosus and 65.5 per cent of the cultures of L. fermenti were sensitive to one or more such bacteriocins. The cultures of L. Fermenti, L. brevis, L. buchneri and L. cellobiosus were sensitive respectively to 37, 19, 16 and 9 types of the bacteriocins out of 39 types tested.     

Lactobacillus plantarum and Lactobacillus buchneri as Expression Systems: Evaluation of Different Origins of Replication for the Design of Suitable Shuttle Vectors

The objectives of this study were to establish transformation protocols for Lactobacillus plantarum CD033 and Lactobacillus buchneri CD034, two industrial silage strains and to test the influence of selected origins of replication on plasmid copy number, plasmid stability, and plasmid incompatibility in these strains. Electro-transformation protocols were optimized by examination of the influence of different electroporation solutions and cell wall weakening agents on transformation efficiency. Using Lithium acetate as cell wall weakening agent, we could achieve transformation efficiencies of 8?×?10(4) transformants per 1?μg DNA for L. buchneri CD034 which is to our knowledge the highest described for this species up to now. In order to test feasibility of previously described origins of replication derived from Bacillus subtilis, L. plantarum, Lactococcus lactis, and two novel L. buchneri CD034 plasmids to drive replication in our two selected Lactobacillus strains, six shuttle vectors were constructed. Results indicate that, in terms of stable propagation and high gene copy numbers (up to 238 copies/chromosome), the most suitable origins of replication for the construction of expression vectors for the selected silage strains were the ones derived from the novel L. buchneri CD034 plasmids.     

The use of a quantitative real-time polymerase chain reaction assay for identification and enumeration of Lactobacillus buchneri in silage

Aims:  To detect and quantify Lactobacillus buchneri in plant samples with the aid of polymerase chain reaction (PCR) methods.
Methods and Results:  DNA from silage samples spiked with different amounts of L. buchneri cells was isolated using a lysozyme/sodium dodecyl sulfate lysis and phenol/chloroform extraction method. The DNA served as a template for PCR amplification with primers specific for the bacterium. The primers were developed by comparison of 16S rDNA sequences from different lactic acid bacteria (LAB) and testing for specificity with 11 different strains of LAB. As few as 100 L. buchneri colony-forming units per gram of silage could be detected. Additionally, the technique was successfully applied to quantify the population of L. buchneri in two cultivars of corn with or without inoculation.
Conclusions:  The PCR assay provided a specific and rapid tool for identifying and enumerating L. buchneri in silage samples.
Significance and Impact of the Study:  The use of microbial inoculants for silage production is a safe and environment friendly practice, but the full potential of such additives can only be achieved with a better understanding of the fate and activity of the microbes involved. The current study describes a methodology to detect and enumerate L. buchneri , a micro-organism used as an inoculant.     

Penicillin-binding proteins of various strains of Lactobacillus

Sensitivity of different species of Lactobacillus i.e. L. casei, L. plantarum, L. acidophillus, L. buchneri, L. jugurti and others to penicillins and cephalosporins of various generations was studied. Penicillin binding proteins (PBPs) of the Lactobacillus species were specified. It was shown that the number of PBPs depended on the Lactobacillus species. L. casei had the least number of PBPs (4) and L. brevis had the highest number of PBPs (11). Competition of 14C-benzylpenicillin with ampicillin, cefotaxime, ceftizoxime and cefoperazone for binding to separate PBPs in three strains of different Lactobacillus species was investigated.     

Bacteriocin typing of some Lactobacillus species of the subgenus betabacterium

One hundred and forty cultures of L. fermenti, L. brevis, and L. buchneri were tested by the method of delayed antagonism for sensitivity to 39 bacteriocins produced by lactobacillus strains of different species. According to their bacteriocin sensitivity patterns, 84 L. fermenti, 43 L. brevis and 13 L. buchneri cultures were differentiated into 26, 18 and 10 bacteriocin types, respectively. Bacteriocin typing allows not only intraspecific differentiation of L. fermenti, L. brevis and L. buchneri cultures but also a subdivision of their biochemical-physiological variants.     

Ribotyping of lactobacilli isolated from spoiled beer

Twenty-nine Lactobacillus strains contaminating beers in different Czech breweries as well as representative type strains obtained from the Czech Collection of Microorganisms were characterized using ribotyping with EcoRI and a probe made complementary to 16S and 23S rRNA genes. Biochemical test results assigned the 29 strains to the species L. brevis, L. plantarum, L. buchneri and L. paracasei subsp. paracasei. Ribotyping separated L. brevis, L. plantarum and L. paracasei subsp. paracasei strains into species-specific ribogroups in full correspondence with biotyping; L. buchneri strains were split into two ribogroups. Characteristic band patterns for each species and even typical bands of certain sizes were observed.     

1,3-Propanediol:NAD+ oxidoreductases of Lactobacillus brevis and Lactobacillus buchneri.

In the cofermentation of glycerol with a sugar by Lactobacillus brevis and Lactobacillus buchneri, a 1,3-propanediol:NAD+ oxidoreductase provides an additional method of NADH disposal. The enzyme has been purified from both L. brevis B22 and L. buchneri B190 and found to have properties very similar to those reported for the enzyme from Klebsiella pneumoniae. The enzymes required Mn2+ and are probably octamers with a molecular mass of 350 kDa. Although not absolutely specific for 1,3-propanediol when tested as dehydrogenases, the enzymes have less than 10% activity with glycerol, ethanol, and 1,2-propanediol. These properties contrast sharply with those of a protein isolated from another Lactobacillus species (L. reuteri) that ferments glycerol with glucose and previously designated a 1,3-propanediol dehydrogenase.     

TEASIng apart alien species risk assessments: a framework for best practices

Some alien species cause substantial impacts, yet most are innocuous. Given limited resources, forecasting risks from alien species will help prioritise management. Given that risk assessment (RA) approaches vary widely, a synthesis is timely to highlight best practices. We reviewed quantitative and scoring RAs, integrating > 300 publications into arguably the most rigorous quantitative RA framework currently existing, and mapping each study onto our framework, which combines Transport, Establishment, Abundance, Spread and Impact (TEASI). Quantitative models generally measured single risk components (78% of studies), often focusing on Establishment alone (79%). Although dominant in academia, quantitative RAs are underused in policy, and should be made more accessible. Accommodating heterogeneous limited data, combining across risk components, and developing generalised RAs across species, space and time without requiring new models for each species may increase attractiveness for policy applications. Comparatively, scoring approaches covered more risk components (50% examined > 3 components), with Impact being the most common component (87%), and have been widely applied in policy (> 57%), but primarily employed expert opinion. Our framework provides guidance for questions asked, combining scores and other improvements. Our risk framework need not be completely parameterised to be informative, but instead identifies opportunities for improvement in alien species RA.     

1,3-Propanediol:NAD+ oxidoreductases of Lactobacillus brevis and Lactobacillus buchneri.

In the cofermentation of glycerol with a sugar by Lactobacillus brevis and Lactobacillus buchneri, a 1,3-propanediol:NAD+ oxidoreductase provides an additional method of NADH disposal. The enzyme has been purified from both L. brevis B22 and L. buchneri B190 and found to have properties very similar to those reported for the enzyme from Klebsiella pneumoniae. The enzymes required Mn2+ and are probably octamers with a molecular mass of 350 kDa. Although not absolutely specific for 1,3-propanediol when tested as dehydrogenases, the enzymes have less than 10% activity with glycerol, ethanol, and 1,2-propanediol. These properties contrast sharply with those of a protein isolated from another Lactobacillus species (L. reuteri) that ferments glycerol with glucose and previously designated a 1,3-propanediol dehydrogenase.     

Alcoholic fermentation and its prevention by Lactobacillus buchneri in whole crop rice silage

AIMS: To evaluate the fermentation characteristics and the effects of Lactobacillus buchneri inoculation in ensiling whole crop rice. METHODS AND RESULTS: Laboratory-scale silages were prepared from whole crop rice harvested at yellow-ripe stage. The crop was ensiled for 2 months with and without inoculation of L. buchneri at 10(4), 10(5) and 10(6) CFU g(-1). The effect of prolonged ensiling was also studied by using the same crop; the silos were opened at 1, 3, 6 and 12 months, while the inoculation was made at 10(5) CFU g(-1). Enhanced alcoholic fermentation was found in untreated silage; the sum of ethanol and 2,3-butanediol were seven times higher at 2 months than those of lactic and volatile fatty acids, while the differences were diminished at 12 months owing to the reduction of ethanol in the late ensiling period. Inoculation of L. buchneri inhibited the alcohols; however, ethanol yet prevailed over the fermentation until 6 months, after which acetic acid became the main product in the inoculated silage. Regardless of inoculation and ensiling period, yeasts were not found in whole crop rice silage. CONCLUSIONS: Substantial amounts of ethanol and 2,3-butanediol would be produced in silage prepared from whole crop rice. The alcoholic fermentation can be suppressed when inoculated with L. buchneri. SIGNIFICANCE AND IMPACT OF THE STUDY: Inoculation of L. buchneri could be an option to prevent ethanol fermentation in silage.     

Coupling mitochondrial respiratory chain to cell death: an essential role of mitochondrial complex I in the interferon-beta and retinoic acid-induced cancer cell death

Combination of retinoic acids (RAs) and interferons (IFNs) has synergistic apoptotic effects and is used in cancer treatment. However, the underlying mechanisms remain unknown. Here, we demonstrate that mitochondrial respiratory chain (MRC) plays an essential role in the IFN-beta/RA-induced cancer cell death. We found that IFN-beta/RA upregulates the expression of MRC complex subunits. Mitochondrial-nuclear translocation of these subunits was not observed, but overproduction of reactive oxygen species (ROS), which causes loss of mitochondrial function, was detected upon IFN-beta/RA treatment. Knockdown of GRIM-19 (gene associated with retinoid-interferon-induced mortality-19) and NDUFS3 (NADH dehydrogenase (ubiquinone) Fe-S protein 3), two subunits of MRC complex I, by siRNA in two cancer cell lines conferred resistance to IFN-beta/RA-induced apoptosis and reduced ROS production. In parallel, expression of late genes induced by IFN-beta/RA that are directly involved in growth inhibition and cell death was also repressed in the knockdown cells. Our data suggest that the MRC regulates IFN-beta/RA-induced cell death by modulating ROS production and late gene expression.     

Anaerobic conversion of lactic acid to acetic acid and 1, 2-propanediol by Lactobacillus buchneri

The degradation of lactic acid under anoxic conditions was studied in several strains of Lactobacillus buchneri and in close relatives such as Lactobacillus parabuchneri, Lactobacillus kefir, and Lactobacillus hilgardii. Of these lactobacilli, L. buchneri and L. parabuchneri were able to degrade lactic acid under anoxic conditions, without requiring an external electron acceptor. Each mole of lactic acid was converted into approximately 0.5 mol of acetic acid, 0.5 mol of 1,2-propanediol, and traces of ethanol. Based on stoichiometry studies and the high levels of NAD-linked 1, 2-propanediol-dependent oxidoreductase (530 to 790 nmol min(-1) mg of protein(-1)), a novel pathway for anaerobic lactic acid degradation is proposed. The anaerobic degradation of lactic acid by L. buchneri does not support cell growth and is pH dependent. Acidic conditions are needed to induce the lactic-acid-degrading capacity of the cells and to maintain the lactic-acid-degrading activity. At a pH above 5.8 hardly any lactic acid degradation was observed. The exact function of anaerobic lactic acid degradation by L. buchneri is not certain, but some results indicate that it plays a role in maintaining cell viability.     

PM2RA: A Framework for Detecting and Quantifying Relationship Alterations in Microbial Community

The dysbiosis of gut microbiota is associated with the pathogenesis of human diseases.However, observing shifts in the microbe abundance cannot fully reveal underlying perturbations.Examining the relationship alterations(RAs) in the microbiome between health and disease statuses provides additional hints about the pathogenesis of human diseases, but no methods were designed to detect and quantify the RAs between different conditions directly. Here, we present profile monitoring for microbial relationship alteration(PM2 RA), an analysis framework to identify and quantify the microbial RAs. The performance of PM2 RA was evaluated with synthetic data, and it showed higher specificity and sensitivity than the co-occurrence-based methods. Analyses of real microbial datasets showed that PM2 RA was robust for quantifying microbial RAs across different datasets in several diseases. By applying PM2 RA, we identified several novel or previously reported microbes implicated in multiple diseases. PM2 RA is now implemented as a web-based application available at http://www.pm2 ra-xingyinliulab.cn/.