Osmotic Fragility of Lysozyme- and Thiol-Treated Lactobacillus plantarum

Reduced but not oxidized thiols increased the sensitivity of lysozyme-treated cells of Lactobacillus plantarum to lysis by osmotic shock.     

Osmotic regulation of intracellular solute pools in Lactobacillus plantarum.

Bacteria respond to changes in medium osmolarity by varying the concentrations of specific solutes in order to maintain constant turgor pressure. The cytoplasmic pools of K+, proline, glutamate, alanine, and glycine of Lactobacillus plantarum ATCC 14917 increased when the osmolarity of the growth media was raised from 0.20 to 1.51 osmol/kg by KCL. When glycine-betaine was present in a high-osmolarity chemically defined medium, it was accumulated to a high cytoplasmic concentration, while the concentrations of most other osmotically important solutes decreased. These observations, together with the effects of glycine-betaine on the specific growth rate under high-osmolarity conditions, suggest that glycine-betaine is preferentially accumulated in L. plantarum. Uptake of glycine-betaine, proline, glutamate, and alanine was studied in cells that were alternately exposed to hyper- and hypo-osmotic stresses. The rate of uptake of proline and glycine-betaine increased instantaneously upon increasing the osmolarity, whereas that of other amino acids did not. This activation occurred also under conditions in which protein synthesis was inhibited was most pronounced when cells were pregrown at high osmolarity. The duration of net transport was a function of the osmotic strength of the assay medium. Glutamate uptake was not activated by an osmotic upshock, and the uptake of alanine was low under all conditions tested. When cells were subjected to osmotic downshock, a rapid efflux of accumulated glycine-betaine, proline, and alanine occurred whereas the pools of other amin acids remained unaffected. The results indicate that osmolyte efflux is, at least to some extent, mediated via specific osmotically regulated efflux systems and not via nonspecific mechanisms as has been suggested previously.     

Mechanism of dehydration inactivation of Lactobacillus plantarum

The mechanism of dehydration inactivation of Lactobacillus plantarum cells after vacuum-drying above saturated salt solutions was studied. The method used is based on the hypothesis that DNase diffuses into cells with damaged cell membranes/walls and hydrolyses the intracellular DNA. Intact, undamaged cells and cells inactivated by either dehydration or heat treatent were incubated in the presence of DNase. The release of DNA hydrolysis products into the incubation medium was measured. It was shown that dehydration inactivation of L. plantarum, but not thermal inactivation, was associated with clear evidence of membrane damage. The residual glucose-fermenting activity of the dehydrated cells related to the release of hydrolysed DNA in the medium, but there was no such relationship with heat-treated cells. Addition of sorbitol to cells before dehydration increased the residual glucose-fermenting activity after drying and this was associated with a reduced rate of DNA hydrolysis. It is concluded that cell wall and/or cell membrane damage is an important mechanism of dehydration inactivation, but that thermal inactivation (up to 60°C) occurs by a different mechanism.Correspondence to: K. van't Riet     

Effects of a Quaternary Ammonium Compound on Escherichia coli

Increasing amounts of tetradecyldimethylbenzyl ammonium chloride (TAC) were lethal to an increasing proportion of an actively growing culture of Escherichia coli. The loss of nucleic acid material by actively growing E. coli did not appear to play a major role in the lethal effect. It was found that lag-phase cells were more sensitive than logarithmic-phase cells to the lethal effect of TAC. The effect of TAC on the lysozyme sensitivity of the test organism was compared with that obtained using disodium dihydrogen ethylenediaminetetraacetate (EDTA). Although TAC was found to render the test organism susceptible to lysozyme, the degree of lysis never reached that attained with EDTA.     

Mechanism of elementary catalytic steps of pyruvate oxidase from Lactobacillus plantarum

Single steps in the catalytic cycle of pyruvate oxidase from Lactobacillus plantarum have been characterized kinetically and mechanistically by stopped-flow in combination with kinetic solvent isotope effect studies. Reversible substrate binding of pyruvate occurs with an on-rate of 6.5 x 10(4) M(-1) s(-1) and an off-rate of pyruvate of 20 s(-1). Decarboxylation of the intermediate lactyl-ThDP and the reduction of FAD which consists of two consecutive single electron-transfer steps from HEThDP to FAD occur with rates of about k(dec) = 112 s(-1) and k(red) = 422 s(-1). Flavin radical intermediates are not observed during reduction, and kinetic solvent isotope effects are absent, indicating that electron transfer and protonation processes are not rate limiting in the overall reduction process. Reoxidation of FADH(2) by O(2) to yield H(2)O(2) takes place at a pseudo-first-order rate of about 35 s(-1) in air-saturated buffer. A comparable value of about 35 s(-1) was estimated for the phosphorolysis of the acetyl-ThDP intermediate at phosphate saturation. In competition with phosphorolysis, enzyme-bound acetyl-ThDP is hydrolyzed with a rate k = 0.03 s(-1). This is the first report in which the reaction of enzyme-bound acetyl-ThDP with phosphate and OH(-) is monitored directly by FAD absorbance changes using the sequential stopped-flow technique.     

Conjugal plasmid transfer (pAM beta 1) in Lactobacillus plantarum

The streptococcal plasmid pAM beta 1 (erythromycin resistance) was transferred via conjugation from Streptococcus faecalis to Lactobacillus plantarum and was transferred among L. plantarum strains. Streptococcus sanguis Challis was transformed with pAM beta 1 isolated from these transconjugants, and transformants harboring intact pAM beta 1 could conjugate the plasmid back to L. plantarum.     

Conjugal plasmid transfer (pAM beta 1) in Lactobacillus plantarum.

The streptococcal plasmid pAM beta 1 (erythromycin resistance) was transferred via conjugation from Streptococcus faecalis to Lactobacillus plantarum and was transferred among L. plantarum strains. Streptococcus sanguis Challis was transformed with pAM beta 1 isolated from these transconjugants, and transformants harboring intact pAM beta 1 could conjugate the plasmid back to L. plantarum.     

Dissecting the Molecular Mechanism of Nucleotide-Dependent Activation of the KtrAB K+ Transporter

KtrAB belongs to the Trk/Ktr/HKT superfamily of monovalent cation (K⁺ and Na⁺) transport proteins that closely resemble K⁺ channels. These proteins underlie a plethora of cellular functions that are crucial for environmental adaptation in plants, fungi, archaea, and bacteria. The activation mechanism of the Trk/Ktr/HKT proteins remains unknown. It has been shown that ATP stimulates the activity of KtrAB while ADP does not. Here, we present X-ray structural information on the KtrAB complex with bound ADP. A comparison with the KtrAB-ATP structure reveals conformational changes in the ring and in the membrane protein. In combination with a biochemical and functional analysis, we uncover how ligand-dependent changes in the KtrA ring are propagated to the KtrB membrane protein and conclude that, despite their structural similarity, the activation mechanism of KtrAB is markedly different from the activation mechanism of K⁺ channels.     

Genome sequence of the naturally plasmid-free Lactobacillus plantarum strain NC8 (CCUG 61730)

Lactobacillus plantarum is a highly versatile lactic acid bacterium found in various ecological niches, such as fermented vegetable, meat, and dairy products and the gastrointestinal tract. We sequenced the genome of L. plantarum NC8, a naturally plasmid-free strain, which has been used as a model strain in many laboratories worldwide.     

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.     

Randomly amplified polymorphic DNA (RAPD) for rapid typing of Lactobacillus plantarum strains

Randomly amplified polymorphic DNA (RAPD) has been used for rapid typing of Lactobacillus plantarum strains. RAPD was used with either purified chromosomal DNA serving as template in the polymerase chain reaction, or with crude cell extracts, and using a 9-mer primer with 80% G + C content. Amplified DNA was visualized by ethidium bromide staining after separation on agarose gels. Patterns from 20 Lact. plantarum strains and two Lact. pentosus strains were analysed using the Pearson products moment correlation coefficient ( r ) and the unweighted pair group method with arithmetic averages (UPGMA). With some exceptions, the two sources of template DNA gave the same clusters and subclusters of strains at the similarity level of 50%. About 50% of the strains could be individually separated from all the other tested strains. The buffer brand, the amount of primer and crude cell extract used in the PCR-step were crucial for the final pattern.     

一株植物乳酸杆菌的高密度发酵

目的研究一株植物乳酸杆菌的高密度发酵。方法通过单因素实验来确定此植物乳酸杆菌的最适生长温度、pH、碳源、氮源、缓冲盐等。结果最适生长温度是37℃、起始pH6.4、最适碳源为葡萄糖、最适氮源为牛肉膏,通过对缓冲盐的选择最终确定NaAc：CaCO3比为0.5：1.5;控制发酵条件,结合优化培养基,可使发酵后的培养液达到2.75×10^10CFU/ml。结论该缓冲盐能够有效的缓解发酵液pH的下降,延长对数生长期,使活菌数提高1倍,适合高密度发酵培养。     

植物乳杆菌CMCC-P0002代谢产物中抑菌物质的研究

目的通过对一株植物乳杆菌代谢产物的抑菌作用以及该产物的某些物理特性的研究,为进一步发现可替代现有抗生素的抗菌物质奠定基础。方法首先利用低温离心和超滤法对植物乳杆菌培养后的上清液进行初步提取,用打孔法行抑菌试验,明确其对铜绿假单胞菌、大肠埃希菌、肺炎克雷伯菌和金黄色葡萄球菌等临床分离菌株的抑菌活性;并进一步经加热、调整pH及过氧化氢酶等处理上清液后,再验证其活性变化。结果植物乳杆菌的培养上清液对铜绿假单胞菌等多株临床分离菌株均具有一定的抑菌活性,且以pH在6以内时该物质抑菌活性较好,过氧化氢酶处理后或加热至100℃、30min,上清液的抑菌活性依然存在。结论在植物乳杆菌的培养上清液中存在着具有抑菌活性的物质,对从临床标本所分离的部分革兰阴性菌及革兰阳性菌有抑菌作用,该物质对热耐受,其活性受pH变化的影响。