Genetic analysis of two bile salt hydrolase activities in Lactobacillus acidophilus NCFM

Two genes, bshA and bshB, encoding bile salt hydrolase enzymes (EC 3.5.1.24) were identified in the genome sequence of Lactobacillus acidophilus NCFM. Targeted inactivation of these genes via chromosomal insertion of an integration vector demonstrated different substrate specificities for these two enzymes.     

Directed chromosomal integration and expression of the reporter gene gusA3 in Lactobacillus acidophilus NCFM

Lactobacillus acidophilus NCFM is a probiotic microbe that survives passage through the human gastrointestinal tract and interacts with the host epithelium and mucosal immune cells. The potential for L. acidophilus to express antigens at mucosal surfaces has been investigated with various antigens and plasmid expression vectors. Plasmid instability and antibiotic selection complicate the possibility of testing these constructs in human clinical trials. Integrating antigen encoding genes into the chromosome for expression is expected to eliminate selection requirements and provide genetic stability. In this work, a reporter gene encoding a β-glucuronidase (GusA3) was integrated into four intergenic chromosomal locations. The integrants were tested for genetic stability and GusA3 activity. Two locations were selected for insertion downstream of constitutively highly expressed genes, one downstream of slpA (LBA0169), encoding a highly expressed surface-layer protein, and one downstream of phosphopyruvate hydratase (LBA0889), a highly expressed gene with homologs in other lactic acid bacteria. An inducible location was selected downstream of lacZ (LBA1462), encoding a β-galactosidase. A fourth location was selected in a low-expression region. The expression of gusA3 was evaluated from each location by measuring GusA3 activity on 4-methyl-umbelliferyl-β-d-glucuronide (MUG). GusA3 activity from both highly expressed loci was more than three logs higher than the gusA3-negative parent, L. acidophilus NCK1909. GusA3 activity from the lacZ locus was one log higher in cells grown in lactose than in glucose. The differences in expression levels between integration locations highlights the importance of rational targeting with gene cassettes intended for chromosomal expression.     

16S ribosomal DNA analysis of the faecal lactobacilli composition of human subjects consuming a probiotic strain Lactobacillus acidophilus NCFM

AIMS: The aims of this study were to evaluate the ability of exogenous Lactobacillus acidophilus strain NCFM to survive through the human gastro-intestinal (GI) tract, and to evaluate the selectivity of Rogosa SL medium for faecal lactobacilli. METHODS AND RESULTS: The composition of the faecal lactobacilli of 10 healthy subjects was monitored for two weeks prior to, two weeks during and two weeks after the administration of the Lact. acidophilus strain NCFM consumed with skim milk (daily dose 10(10) viable cells). Fresh faecal samples were collected, processed and cultured on Rogosa SL selective medium for lactobacilli enumeration. Colonies demonstrating various morphologies were identified and purified for 16S ribosomal DNA sequence analysis for speciation of colonial genotype. The species composition of cultivable faecal lactobacilli changed considerably during consumption of the strain NCFM. CONCLUSIONS: The probiotic Lact. acidophilus strain NCFM can survive through the human GI tract, but cannot colonize itself during the two-week consumption. Rogosa SL medium is selective for faecal lactobacilli. However, genetic analysis is required for colony speciation. SIGNIFICANCE AND IMPACT OF THE STUDY: It is demonstrated that continuous consumption is necessary to maintain a high population of the probiotic strain, and that the Rogosa SL medium is reliable.     

Lactobacillus helveticus MIMLh5-Specific Antibodies for Detection of S-Layer Protein in Grana Padano Protected-Designation-of-Origin Cheese

Single-chain variable-fragment antibodies (scFvs) have considerable potential in immunological detection and localization of bacterial surface structures. In this study, synthetic phage-displayed antibody libraries were used to select scFvs against immunologically active S-layer protein of Lactobacillus helveticus MIMLh5. After three rounds of panning, five relevant phage clones were obtained, of which four were specific for the S-layer protein of L. helveticus MIMLh5 and one was also capable of binding to the S-layer protein of L. helveticus ATCC 15009. All five anti-S-layer scFvs were expressed in Escherichia coli XL1-Blue, and their specificity profiles were characterized by Western blotting. The anti-S-layer scFv PolyH4, with the highest specificity for the S-layer protein of L. helveticus MIMLh5, was used to detect the S-layer protein in Grana Padano protected-designation-of-origin (PDO) cheese extracts by Western blotting. These results showed promising applications of this monoclonal antibody for the detection of immunomodulatory S-layer protein in dairy (and dairy-based) foods.     

Efficient System for Directed Integration into the Lactobacillus acidophilus and Lactobacillus gasseri Chromosomes via Homologous Recombination

An efficient method is described for the generation of site-specific chromosomal integrations in Lactobacillus acidophilus and Lactobacillus gasseri. The strategy is an adaptation of the lactococcal pORI system (K. Leenhouts, G. Venema, and J. Kok, Methods Cell Sci. 20:35–50, 1998) and relies on the simultaneous use of two plasmids. The functionality of the integration strategy was demonstated by the insertional inactivation of the Lactobacillus acidophilus NCFM lacL gene encoding β-galactosidase and of the Lactobacillus gasseri ADH gusA gene encoding β-glucuronidase.     

摄入含嗜酸乳杆菌NCFM和乳双歧杆菌Bi-07的益生菌补充剂增强免疫功能的动物实验研究

目的测定与验证摄入含特定乳酸菌株组合（嗜酸乳杆菌NCFM和乳双歧杆菌Bi-07）一定剂量的益生菌补充剂对动物（小鼠）免疫功能的影响。方法SPF昆明种小白鼠经口连续分别给予0．25、0．50、1．50g／kgBW的益生菌补充剂4周,进行迟发型变态反应试验、溶血素滴度测定、NK细胞活性测定等。结果在小白鼠迟发型变态反应试验中,中、高剂量组足跖增厚值均高于对照组,差异有显著性（P〈0．05）;对受试动物血清溶血素抗体滴度水平的影响的实验中,中、高剂量组的抗体积数高于对照组,差异均有显著性（P〈0．05）。NK细胞活性测定中,高剂量组脾NK细胞活性增强,差异有非常显著性（P〈0．01）。结论含该两种特定乳酸菌株的益生菌补充剂被小自鼠摄入一定剂量后,起到了增强小白鼠细胞免疫、体液免疫功能及NK细胞活性的作用。     

嗜酸乳杆菌LA-G80的毒理学研究

目的对嗜酸乳杆菌的毒性进行研究。方法采用大、小鼠急性毒性试验、Ames试验、小鼠骨髓细胞微核试验、小鼠精子畸形试验和大鼠30 d喂养等对嗜酸乳杆菌进行安全性试验研究。结果急性经口毒性试验表明,大、小鼠灌胃给予嗜酸乳杆菌,最大耐受剂量雌雄两性别均大于20.0 g/kg体重,Ames试验、微核试验和精子畸形试验结果均为阴性。大鼠30 d喂养试验结果表明各项指标均未见明显毒性反应。结论在本次实验条件下,嗜酸乳杆菌未见遗传毒性。由此可初步判定,使用嗜酸乳杆菌是安全可靠的。     

16S ribosomal DNA terminal restriction fragment pattern analysis of bacterial communities in feces of rats fed Lactobacillus acidophilus NCFM

16S ribosomal DNA terminal restriction fragment patterns from rat fecal samples were analyzed to track the dynamics of Lactobacillus acidophilus NCFM and discern bacterial populations that changed during feeding with NCFM. Lactobacillus johnsonii and Ruminococcus flavefaciens were tentatively identified as such bacterial populations. The presence of L. johnsonii was confirmed by isolation from feces.     

16S Ribosomal DNA Terminal Restriction Fragment Pattern Analysis of Bacterial Communities in Feces of Rats Fed Lactobacillus acidophilus NCFM

16S ribosomal DNA terminal restriction fragment patterns from rat fecal samples were analyzed to track the dynamics of Lactobacillus acidophilus NCFM and discern bacterial populations that changed during feeding with NCFM. Lactobacillus johnsonii and Ruminococcus flavefaciens were tentatively identified as such bacterial populations. The presence of L. johnsonii was confirmed by isolation from feces.     

嗜酸乳杆菌固态发酵的初步研究

研究了嗜酸乳杆菌在 4种不同固态基质 (豆渣、麦麸、芋艿渣、纤维素 )中的生长情况 ,结果显示 :1)豆渣作为基质时嗜酸乳杆菌生长最佳 ,最高活菌数可达 1.6 45× 10 9cfu/g ,麦麸次之 ,达 3.2× 10 8cfu/g ,纤维素最差 ,仅为 2 .1× 10 7cfu/g ,并且豆渣固态培养基优于对照的 3种液体培养基 ;2 )豆渣的含水量以 75 %左右为宜 ;3)通过加入可中和所产酸量的CaCO3 ,并用缓冲液代替水配制培养基 ,结果发现用pH 6 .0的缓冲液配制的培养基 ,培养后活菌量可达 2 .12 4× 10 9cfu/g ,,比对照用自来水提高了 4.2倍 .     

Characterization of Rhamnosidases from Lactobacillus plantarum and Lactobacillus acidophilus

Lactobacilli are known to use plant materials as a food source. Many such materials are rich in rhamnose-containing polyphenols, and thus it can be anticipated that lactobacilli will contain rhamnosidases. Therefore, genome sequences of food-grade lactobacilli were screened for putative rhamnosidases. In the genome of Lactobacillus plantarum, two putative rhamnosidase genes (ram1_Lp and ram2_Lp) were identified, while in Lactobacillus acidophilus, one rhamnosidase gene was found (ramA_La). Gene products from all three genes were produced after introduction into Escherichia coli and were then tested for their enzymatic properties. Ram1_Lp, Ram2_Lp, and RamA_La were able to efficiently hydrolyze rutin and other rutinosides, while RamA_La was, in addition, able to cleave naringin, a neohesperidoside. Subsequently, the potential application of Lactobacillus rhamnosidases in food processing was investigated using a single matrix, tomato pulp. Recombinant Ram1_Lp and RamA_La enzymes were shown to remove the rhamnose from rutinosides in this material, but efficient conversion required adjustment of the tomato pulp to pH 6. The potential of Ram1_Lp for fermentation of plant flavonoids was further investigated by expression in the food-grade bacterium Lactococcus lactis. This system was used for fermentation of tomato pulp, with the aim of improving the bioavailability of flavonoids in processed tomato products. While import of flavonoids into L. lactis appeared to be a limiting factor, rhamnose removal was confirmed, indicating that rhamnosidase-producing bacteria may find commercial application, depending on the technological properties of the strains and enzymes.Lactobacilli such as Lactobacillus plantarum have been used for centuries to ferment vegetables such as cabbage, cucumber, and soybean (34). Fruit pulps, for instance, those from tomato, have also been used as a substrate for lactobacilli for the production of probiotic juices (38). Recently, the full genomic sequences of several lactobacilli have become available (1, 22). A number of the plant-based substrates for lactobacilli are rich in rhamnose sugars, which are often conjugated to polyphenols, as in the case of cell wall components and certain flavonoid antioxidants. Utilization of these compounds by lactobacilli would involve α-l-rhamnosidases, which catalyze the hydrolytic release of rhamnose. Plant-pathogenic fungi such as Aspergillus species produce the rhamnosidases when cultured in the presence of naringin, a rhamnosilated flavonoid (24, 26). Bacteria such as Bacillus species have also been shown to use similar enzyme activities for metabolizing bacterial biofilms which contain rhamnose (17, 40).In food processing, rhamnosidases have been applied primarily for debittering of citrus juices. Part of the bitter taste of citrus is caused by naringin (Fig. ​(Fig.1),1), which loses its bitter taste upon removal of the rhamnose (32). More recently, application of rhamnosidases for improving the bioavailability of flavonoids has been described. Human intake of flavonoids has been associated with a reduced risk of coronary heart disease in epidemiological studies (19). Food flavonoids need to be absorbed efficiently from what we eat in order to execute any beneficial function. Absorption occurs primarily in the small intestine (12, 37). Unabsorbed flavonoids will arrive in the colon, where they will be catabolized by the microflora, which is then present in huge quantities. Therefore, it would be desirable for flavonoids to be consumed in a form that is already optimal for absorption in the small intestine prior to their potential degradation. For the flavonoid quercetin, it has been demonstrated that the presence of rhamnoside groups inhibits its absorption about fivefold (20). A number of flavonoids which are present in frequently consumed food commodities, such as tomato and citrus products, often carry rutinoside (6-β-l-rhamnosyl-d-glucose) or neohesperidoside (2-β-l-rhamnosyl-d-glucose) residues (Fig. ​(Fig.1).1). Therefore, removal of the rhamnose groups from such flavonoid rutinosides and neohesperidosides prior to consumption could enhance their intestinal absorption. With this aim, studies were recently carried out toward the application of fungal enzyme preparations as a potential means to selectively remove rhamnoside moieties (16, 30).Open in a separate windowFIG. 1.Chemical structures of rhamnose-containing flavonoids from plants. Relevant carbon atoms in glycoside moieties are numbered. (1) Rutin (quercetin-3-glucoside-1→6-rhamnoside); (2) narirutin (naringenin-7-glucoside-1→6-rhamnoside); (3) naringin (naringenin-7-glucoside-1→2-rhamnoside); (4) p-nitrophenol-rhamnose.In view of the frequent occurrence of lactobacilli on decaying plant material and fermented vegetable substrates, one could anticipate that their genomes carry one or more genes encoding enzymes capable of utilizing rhamnosilated compounds. In the work reported here, we describe the identification of three putative rhamnosidase genes in lactobacillus genomes. We expressed these genes in Escherichia coli and characterized their gene products. The activities of all three lactobacillus rhamnosidases on flavonoids naturally present in tomato pulp were then assessed. One of the L. plantarum genes, which encoded the enzyme with the highest activity and stability in E. coli, was then also expressed in Lactococcus lactis, with the aim of investigating the potential use of such a recombinant organism to improve the bioavailability of fruit flavonoids and thus their efficacy in common foodstuffs.     

嗜酸乳杆菌活菌制剂保护剂及其工艺的研究

为提高嗜酸乳杆菌片剂的活菌存活率,延长其有效期,在制备过程中添加两组保护剂并对其配方进行了优化.首先利用数学统计方法Plackett-Burman设计,对扩大培养时加入的8种材料及制剂压片时的12种材料的保护效果进行评价,分别筛选出影响嗜酸乳杆菌存活率的重要因素;接着用均匀设计法进一步得到这些重要因素的最适百分比.验证试验表明:扩大培养时加入氯化钠0.3％、甘露醇0.4％、麦芽糖3.5％有显著保护作用;制剂压片时加入抗坏血酸1.0％、蔗糖35.0％有显著保护作用.     

Construction of a Gene Knockout System for Application in Paenibacillus alvei CCM 2051T,Exemplified by the S-Layer Glycan Biosynthesis Initiation Enzyme WsfP

The gram-positive bacterium Paenibacillus alvei CCM 2051^T is covered by an oblique surface layer (S-layer) composed of glycoprotein subunits. The S-layer O-glycan is a polymer of [→3)-β-d-Galp-(1[α-d-Glcp-(1→6)]→4)-β-d-ManpNAc-(1→] repeating units that is linked by an adaptor of -[GroA-2→OPO₂→4-β-d-ManpNAc-(1→4)]→3)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-β-d-Galp-(1→ to specific tyrosine residues of the S-layer protein. For elucidation of the mechanism governing S-layer glycan biosynthesis, a gene knockout system using bacterial mobile group II intron-mediated gene disruption was developed. The system is further based on the sgsE S-layer gene promoter of Geobacillus stearothermophilus NRS 2004/3a and on the Geobacillus-Bacillus-Escherichia coli shuttle vector pNW33N. As a target gene, wsfP, encoding a putative UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase, representing the predicted initiation enzyme of S-layer glycan biosynthesis, was disrupted. S-layer protein glycosylation was completely abolished in the insertional P. alvei CCM 2051^T wsfP mutant, according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis evidence and carbohydrate analysis. Glycosylation was fully restored by plasmid-based expression of wsfP in the glycan-deficient P. alvei mutant, confirming that WsfP initiates S-layer protein glycosylation. This is the first report on the successful genetic manipulation of bacterial S-layer protein glycosylation in vivo, including transformation of and heterologous gene expression and gene disruption in the model organism P. alvei CCM 2051^T.Bacterial cell surface layer (S-layer) glycoproteins provide a unique self-assembly matrix that has been optimized by nature for regular and periodic display of glycans with nanometer scale accuracy (21, 31). Exploitation of this self-assembly system for surface display of functional, tailor-made glycans is an attractive alternative to the use of common cell surface anchors (7), with potential areas of application relating to any biological phenomenon that is based on carbohydrate recognition, such as receptor-substrate interaction, signaling, or cell-cell communication. A prerequisite for this endeavor is the availability of an S-layer glycoprotein-covered bacterium that is amenable to genetic manipulation. Despite the high application potential offered by the S-layer glycan display system, there are so far only two reports in the literature dealing with the genetic manipulation of S-layer glycoprotein-carrying bacteria. Both reports concern the gram-negative periodontal pathogen Tannerella forsythia ATCC 43037, but neither of them affects S-layer protein glycosylation (12, 24). In archaea, in contrast, molecular studies of S-layer protein glycosylation are quite advanced (1), but with the archaeal system, S-layer glycoprotein self-assembly, which is a prerequisite for the desired glycan display, has not been manageable in vitro so far.Our model organisms and, hence, candidates for S-layer-mediated glycan display enabled by carbohydrate engineering techniques are members of the Bacillaceae family. Currently, the S-layer glycosylation system of the thermophilic bacterium Geobacillus stearothermophilus NRS 2004/3a is best understood (20, 23, 29, 33, 34). However, a drawback of this organism is its resistance to take up foreign DNA. Although described in the literature (13, 14, 37), transformation of thermophilic bacilli seems to be a strain-specific trait. Based on successful transformation experiments in our laboratory, the mesophilic bacterium Paenibacillus alvei CCM 2051^T (ATCC 6344; DSM 29) (formerly Bacillus alvei [4]) was chosen to set up a system for genetic manipulation. The bacterium is completely covered with an oblique S-layer lattice composed of glycoprotein species. Various aspects of its S-layer, including ultrastructural characterization (27), glycosylation analysis (2, 18), and glycan biosynthesis (11), have been investigated so far. The S-layer O-glycans are polymers of [→3)-β-d-Galp-(1[α-d-Glcp-(1→6)]→4)-β-d-ManpNAc-(1→] repeating units that are linked via the adaptor -[GroA-2→OPO₂→4-β-d-ManpNAc-(1→4)]→3)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-β-d- Galp-(1→ to specific tyrosine residues (2, 18) of the S-layer protein SpaA (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"FJ751775","term_id":"225546017","term_text":"FJ751775"}}FJ751775).Due to the presence of an identical adaptor saccharide backbone in the S-layer glycan of G. stearothermophilus NRS 2004/3a (29), where its biosynthesis is initiated by the UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase WsaP (33), it was conceivable that a homologous enzyme would initiate S-layer glycosylation in P. alvei CCM 2051^T. Considering that the S-layer protein glycosylation machinery has been found to be encoded by S-layer glycosylation (slg) gene clusters (21), degenerate primers for the rml genes catalyzing the dTDP-l-Rha biosynthesis required for building up the adaptor saccharide of the P. alvei CCM 2051^T S-layer glycan were used to define a point of entry into the glycosylation locus (K. Zarschler, B. Janesch, P. Messner, and C. Schäffer, unpublished data). Chromosome walking revealed the existence of an slg gene cluster of about 24 kb, including an open reading frame (ORF) predicted to encode the initiation enzyme of S-layer protein glycosylation. The corresponding gene, named wsfP, served as a first target for the gene knockout system developed in the course of the present study. This target was chosen because loss of function would be easily screenable, resulting in an S-layer glycosylation-deficient mutant. The gene knockout system constructed for insertional inactivation of the chromosomal wsfP gene of P. alvei CCM 2051^T is based on the commercially available bacterial mobile group II intron Ll.LtrB of Lactococcus lactis, in combination with further components available in our laboratory, including the broad-host-range S-layer gene promoter of sgsE from G. stearothermophilus NRS 2004/3a (22) and the Geobacillus-Bacillus-Escherichia coli shuttle vector pNW33N. Bacterial mobile group II introns are retroelements inserted into specific DNA target sites at high frequency by use of the retrohoming mechanism, by which the excised intron lariat RNA is inserted directly into a DNA target site and is then reverse transcribed by the associated intron-encoded enzyme protein (6, 8, 17). Since the DNA target site is recognized primarily by base pairing of intron RNA, which can be modified, and a few intron-encoded-enzyme-protein recognition positions, these introns can be inserted efficiently into any specific DNA target (9, 15, 35, 40).The aim of this study is the development of a genetic tool for manipulation of S-layer protein glycosylation in P. alvei CCM 2051^T. For proof of concept, we specifically deal with (i) the construction of a broad-host-range gene knockout system based on the L. lactis Ll.LtrB intron; (ii) its modification for specific disruption of the wsfP gene on the P. alvei CCM 2051^T chromosome, encoding the putative initiation enzyme of S-layer glycan biosynthesis; and (iii) the reconstitution of enzyme activity by plasmid-based expression of wsfP and its predicted functional homologue wsaP from G. stearothermophilus NRS 2004/3a.     

嗜酸乳杆菌与嗜热链球菌共发酵互生机理的研究

目的:对嗜酸乳杆菌和嗜热链球菌共发酵时两者的互生作用机理进行研究。方法:以质量浓度100gL脱脂乳为培养基,将嗜酸乳杆菌主要代谢产物—氨基酸,如赖氨酸、精氨酸、缬氨酸分别以0.0083mgml、0.0036mgml、0.0053mgml的量加入含嗜热链球菌脱脂乳培养基中,40℃培养,测定凝乳时间;将嗜热链球菌的代谢产物-乳酸、甲酸分别为0.1332mgml和0.075mgml的量加入含嗜酸乳杆菌脱脂乳培养基中,37℃培养,测定其发酵乳的凝乳时间。结果:嗜热链球菌发酵乳凝乳时间由12h缩短到4h,pH为4.52～4.62,吉尔涅尔度为54.23～64.74°T;嗜酸乳杆菌发酵乳的凝乳时间由16h缩短为5h,pH为4.61～4.65;且嗜酸乳杆菌在CO2环境中发酵时,发酵时间明显缩短。结论:嗜酸乳杆菌和嗜热链球菌共发酵时具有互生关系。     

Lactobacillus acidophilus contributes to a healthy environment for vaginal epithelial cells

Lactobacillus species in the female genital tract are thought to act as a barrier to infection. Several studies have demonstrated that lactobacilli can adhere to vaginal epithelial cells. However, little is known about how the adherence of lactobacilli to vaginal epithelial cells affects the acidity, cell viability, or proliferation of the lactobacilli themselves or those of vaginal epithelial cells. Lactobacillus acidophilus was co-cultured with immortalized human vaginal epithelial cells (MS74 cell line), and the growth of L. acidophilus and the acidity of the culture medium were measured. MS74 cell density and viability were also assessed by counting cell numbers and observing the cell attachment state. L. acidophilus showed exponential growth for the first 6 hr until 9 hr, and the pH was maintained close to 4.0-5.0 at 24 hr after culture, consistent with previous studies. The growth curve of L. acidophilus or the pH values were relatively unaffected by co-culture with MS74 cells, confirming that L. acidophilus maintains a low pH in the presence of MS74 cells. This co-culture model could therefore potentially be used to mimic vaginal conditions for future in vitro studies. On the other hand, MS74 cells co-cultured with L. acidophilus more firmly attached to the culture plate, and a higher number of cells were present compared to cells cultured in the absence of L. acidophilus. These results indicate that L. acidophilus increases MS74 cell proliferation and viability, suggesting that lactobacilli may contribute to the healthy environment for vaginal epithelial cells.