Generation of Food-Grade Lactococcal Starters Which Produce the Lantibiotics Lacticin 3147 and Lacticin 481

Transconjugant lactococcal starters which produce both lantibiotics lacticin 3147 and lacticin 481 were generated via conjugation of large bacteriocin-encoding plasmids. A representative of one of the resultant strains proved more effective at killing Lactobacillus fermentum and inhibiting the growth of Listeria monocytogenes LO28H than either of the single bacteriocin-producing parental strains, demonstrating the potential of these transconjugants as protection cultures for food safety applications.     

Overproduction of Wild-Type and Bioengineered Derivatives of the Lantibiotic Lacticin 3147

Lacticin 3147 is a broad-spectrum two-peptide lantibiotic whose genetic determinants are located on two divergent operons on the lactococcal plasmid pMRC01. Here we introduce each of 14 subclones, containing different combinations of lacticin 3147 genes, into MG1363 (pMRC01) and determine that a number of them can facilitate overproduction of the lantibiotic. Based on these studies it is apparent that while the provision of additional copies of genes encoding the biosynthetic/production machinery and the regulator LtnR is a requirement for high-level overproduction, the presence of additional copies of the structural genes (i.e., ltnA1A2) is not.     

Fate of the Two-Component Lantibiotic Lacticin 3147 in the Gastrointestinal Tract

The component peptides of lacticin 3147 were degraded by α-chymotrypsin in vitro with a resultant loss of antimicrobial activity. Activity was also lost in ileum digesta. Following oral ingestion, neither of the lacticin 3147 peptides was detected in the gastric, jejunum, or ileum digesta of pigs, and no lacticin 3147 activity was found in the feces. These observations suggest that lacticin 3147 ingestion is unlikely to have adverse effects, since it is probably inactivated during intestinal transit.     

Lacticin 3147, a Broad-Spectrum Bacteriocin Which Selectively Dissipates the Membrane Potential

Lacticin 3147 is a broad-spectrum bacteriocin produced by Lactococcus lactis subsp. lactis DPC3147 (M. P. Ryan, M. C. Rea, C. Hill, and R. P. Ross, Appl. Environ. Microbiol. 62:612–619, 1996). Partial purification of the bacteriocin by hydrophobic interaction chromatography and reverse-phase fast protein liquid chromatography revealed that two components are required for full activity. Lacticin 3147 is bactericidal against L. lactis, Listeria monocytogenes, and Bacillus subtilis; at low concentrations of the bacteriocin, bactericidal activity is enhanced when target cells are energized. This finding suggests that the presence of a proton motive force promotes the interaction of the bacteriocin with the cytoplasmic membrane, leading to the formation of pores at these low lacticin 3147 concentrations. These pores were shown to be selective for K⁺ ions and inorganic phosphate. The loss of these ions resulted in immediate dissipation of the membrane potential and hydrolysis of internal ATP, leading to an eventual collapse of the pH gradient at the membrane and ultimately to cell death. Our results suggest that lacticin 3147 is a pore-forming bacteriocin which acts on a broad range of gram-positive bacteria.     

乳链菌肽Nisin的生物合成及表达调控机制

乳链菌肽Nisin是乳酸乳球菌（Lactococcus lactis）产生的一种多肽类抗菌物质,是一种由34个氨基酸组成的羊毛硫细菌素。与Nisin合成有关的基因有11个,构成一个基因簇nisA（Z）BTCIPRKFEG。这些相关基因组成三个操纵子进行转录,分别是nisA（Z）BTCIP、nisRK和nisFEG。Nisin通过NisRK双组分调节系统诱导自身合成,而NisI和NisFEG赋予了Nisin产生菌对Nisin的免疫性。对于Nisin的生物合成机制人们展开了非常广泛的研究。本文对Nisin的结构、Nisin合成相关的基因簇、Nisin的生物合成及表达调控机制以及Nisin产生菌对Nisin的免疫性进行了综述。     

Lacticin 3147 favours isoleucine transamination by Lactococcus lactis IFPL359 in a cheese-model system

The bacteriocin, lacticin 3147, increased isoleucine transamination by Lactococcus lactis IFPL359 in a cheese model system. The formation of -keto--methyl-n-valeric acid and 2-hydroxy-3-methyl-valeric acid increased by three times in cheese slurries at 12 °C and cheese aroma intensity increased as well, which corresponded with a higher 2-methylbutanal formation.     

Purification and Characterization of Glutamyl-tRNA Synthetase : An Enzyme Involved in Chlorophyll Biosynthesis

Chlorophyll biosynthesis starts with the synthesis of glutamyl-tRNA (glu-tRNA) by a glutamyl-tRNA synthetase (Glu RS). The glu-tRNA is subsequently transformed to δ-aminolevulinic acid (ALA), which is a committed and regulated precursor in the chlorophyll biosynthetic pathway. The Glu RS from a green alga, Chlamydomonas reinhardtii, was purified and shown to be able to synthesize glu-tRNA and to participate in ALA synthesis in a coupled enzyme assay. Physical and chemical characterization of the purified Glu RS indicated that the enzyme had been purified to homogeneity. The purified enzyme has a native molecular weight of 60,000, an isoelectric point of 4.6, and it formed a single band of 32,500 daltons when analyzed by a silver stained denaturing gel. The N-terminal amino acid sequence of the 32,500 dalton protein was determined to be Asn-Lys-Val-Ala-Leu-Leu-Gly-Ala-Ala-Gly. The molecular weight analyses together with the unambiguous N-terminal amino acid sequence obtained from the purified enzyme suggested that the native enzyme was composed of two identical subunits. Polyclonal antibodies raised against the purified and denatured enzyme were able to inhibit the activity of the native enzyme and to interact specifically with the 32,500 dalton band on Western blots. Thus, the antibodies provided an additional linkage for the structural and functional identities of the enzyme. In vitro experiments showed that over 90% of the glu RS activity was inhibited by 5 micromolar heme, which suggested that Glu RS may be a regulated enzyme in the chlorophyll biosynthetic pathway.     

Some Aspects of the Enzyme Activities Involved in Coenzyme A Biosynthesis in Various Microorganisms

The distribution of the enzyme activities relating to CoA biosynthesis from pantothenic acid in various microorganisms and the effect of CoA on these activities are described.

High activities of partial reactions involved in CoA biosynthesis were surveyed in various type culture strains involving bacteria, actinomycetes, lactic acid bacteria, molds, and yeasts. Generally, higher activities were found in bacteria. CoA inhibited the phosphorylation of pantothenic acid, and resulted in a decrease of CoA production in all the CoA producing strains, while only a little inhibition by CoA was observed in the other reactions, and CoA production from pantothenic acid 4′-phosphate by Brevibacterium ammoniagenes IFO 12071 was not repressed even in the presence of 4mm of CoA. Extracellular excretion of the enzymes of CoA biosynthesis was observed when cells were in contact with sodium lauryl sulfate. Degrading activity against CoA and that against AMP were relatively lower in CoA producing strains when compared with those in other strains. It was confirmed that Brown’s route of CoA biosynthesis operates in Brevibacterium ammoniagenes IFO 12071.     

Strategy for Manipulation of Cheese Flora Using Combinations of Lacticin 3147-Producing and -Resistant Cultures

The aim of the present study was to develop adjunct strains which can grow in the presence of bacteriocin produced by lacticin 3147-producing starters in fermented products such as cheese. A Lactobacillus paracasei subsp. paracasei strain (DPC5336) was isolated from a well-flavored, commercial cheddar cheese and exposed to increasing concentrations (up to 4,100 arbitrary units [AU]/ml) of lantibiotic lacticin 3147. This approach generated a stable, more-resistant variant of the isolate (DPC5337), which was 32 times less sensitive to lacticin 3147 than DPC5336. The performance of DPC5336 was compared to that of DPC5337 as adjunct cultures in two separate trials using either Lactococcus lactis DPC3147 (a natural producer) or L. lactis DPC4275 (a lacticin 3147-producing transconjugant) as the starter. These lacticin 3147-producing starters were previously shown to control adventitious nonstarter lactic acid bacteria in cheddar cheese. Lacticin 3147 was produced and remained stable during ripening, with levels of either 1,280 or 640 AU/g detected after 6 months of ripening. The more-resistant adjunct culture survived and grew in the presence of the bacteriocin in each trial, reaching levels of 10⁷ CFU/g during ripening, in contrast to the sensitive strain, which was present at levels 100- to 1,000-fold lower. Furthermore, randomly amplified polymorphic DNA-PCR was employed to demonstrate that the resistant adjunct strain comprised the dominant microflora in the test cheeses during ripening.     

生物合成乳链菌肽及其螯合物的研究

从酸奶中筛选到一株乳链菌肽产生菌A1-06,研究了各种条件因素对其合成能力的影响。通过发酵培养基的优化,在以质量分数2.0%的蔗糖为唯一碳源、0.25%的酵母膏为唯一氮源条件下,A1-06合成乳链菌肽的产率为0.3 g.L-1,效价为1.018×106U.L-1,比在基础培养基中合成的活性提高17%。Mn2+对A1-06的合成能力有抑制作用,而吐温-80则有促进作用。对乳链菌肽及其Zn2+和Fe2+的螯合物的抑菌效果进行了比较,结果表明,乳链菌肽螯合Fe(Ⅱ)对G-菌有抑制作用,6 h的抑菌率为50.3%,而乳链菌肽、乳链菌肽螯合Zn(Ⅱ)对G-菌无明显的抑制作用。     

刺五加皂苷合成关键酶基因表达的半定量RT-PCR分析

根据已克隆的刺五加鲨烯合酶(squalene synthase,SS)、鲨烯环氧酶(squalene epoxidase,SE)和β-香树酯醇合成酶(β-amyrin synthase,bAS)基因序列信息设计引物,通过半定量RT-PCR分析了SS、SE和bAS基因在刺五加不同生长发育时期和不同器官中表达量的变化.结果表明,SS、SE和bAS基因在各生长发育时期和各器官中均有表达,但表达量差异显著(P<0.05),三者均在盛花期表达量最高,之后降低,进入果实成熟期后SS和bAS的表达量迅速回升,SE无显著变化.SS和bAS在叶片和根中的表达量较高,SE表达量的最大值出现在叶片和幼茎中.刺五加SS、SE和bAS基因的表达间存在显著的正相关关系(P<0.05).研究结果为进一步分析关键酶基因对刺五加三萜皂苷生物合成的影响奠定了基础.     

Lacticin 3147 displays activity in buffer against Gram-positive bacterial pathogens which appear insensitive in standard plate assays

Lacticin 3147 is a broad-spectrum bacteriocin produced by Lactococcus lactis subsp. lactis DPC3147, which has been shown to be active against a range of food-borne bacteria. The reported inhibitory range for lacticin is extended to include methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecalis, penicillin-resistant Pneumococcus, Propionibacterium acne and Streptococcus mutans. This extended host range is not obvious from traditional agar plate-based methods, but reductions in bacterial cell numbers by up to 6 log10 cfu ml-1 was observed after 2 h in time-kill curve studies conducted in broth, suggesting that the bacteriocin may have potential as a therapeutic agent in the treatment of human infections.     

Aldoxime-Forming Microsomal Enzyme Systems Involved in the Biosynthesis of Glucosinolates in Oilseed Rape (Brassica napus) Leaves

Glucosinolates and cyanogenic glucosides are synthesized from amino acids via similar intermediates, N-hydroxyamino acids and aldoximes. Microsomal preparations from young green leaves of oilseed rape catalyze the NADPH-dependent metabolism of homo-phenylalanine and dihomomethionine to the respective aldoximes, precursors of 2-phenylethyl and 3-butenyl glucosinolates. Cytochrome P-450-type enzymes are not involved (in contrast to cyanogenic glucoside biosynthesis), because neither activity was affected by carbon monoxide or other cytochrome P-450 inhibitors. Copper ions and diethyl pyrocarbonate were potent inhibitors of the enzymes, and treatment of microsomes with detergents abolished the overall activity. Two distinct enzyme systems with similar properties appear to be involved, each specific for a particular substrate. One utilizes dihomomethionine and is not active with homophenylalanine or any other amino acid tested, and the other is specific for homophenylalanine. From the characteristics of these enzymes, it seems that these early steps in glucosinolate biosynthesis may be catalyzed by flavin-containing monooxygenases comparable to those found in mammalian tissues and elsewhere. The pathways for the biosynthesis of glucosinolates and cyanogenic glucosides have apparently evolved independently, despite the similar chemical conversions involved.     

Isolation of a Microsomal Enzyme System Involved in Glucosinolate Biosynthesis from Seedlings of Tropaeolum majus L

An in vitro system that converts phenylalanine to phenylacetaldoxime in the biosynthesis of the glucosinolate glucotropaeolin has been established in seedlings of Tropaeolum majus L. exposed to the combined treatment of jasmonic acid, ethanol, and light. The treatment resulted in a 9-fold induction, compared with untreated, dark-grown seedlings, of de novo biosynthesis measured as incorporation of radioactively labeled phenylalanine into glucotropaeolin. Formation of the inhibitory degradation product benzylisothiocyanate during tissue homogenization was prevented by inactivation of the thioglucosidase myrosinase by addition of 100 mM ascorbic acid to the isolation buffer. This allowed the isolation of a biosynthetically active microsomal preparation from the induced T. majus plant material. The enzyme, which catalyzes the conversion of phenylalanine to the corresponding oxime, was sensitive to cytochrome P450 inhibitors, indicating the involvement of a cytochrome P450 in the biosynthetic pathway. It has previously been shown that the oxime-producing enzyme in the biosynthesis of p-hydroxybenzylglucosinolate in Sinapis alba L. is dependent on cytochrome P450, whereas the oxime-producing enzymes in Brassica species have been suggested to be flavin monooxygenases or peroxidase-type enzymes. The result with T. majus provides additional experimental documentation for a similarity between the enzymes converting amino acids into the corresponding oximes in the biosynthesis of glucosinolates and cyanogenic glucosides.     

植物纤维素生物合成及其相关酶类

纤维素是由成千上万个D-葡萄糖分子通过β-1,4糖苷键连接的具有一定立体构象的链状聚合物,其葡萄糖残基约为2000～25000个。它是细胞壁的主要组成成分之一。植物,大多数藻类,一些细菌和真菌甚至有些动物都能合成纤维素。它作为世界上最丰富的,具有巨大商业价值的生物多聚体,几十年来一直受到人们的重视,成为人们的研究热点。尽管如此,这方面的研究仍比较滞后,人们对纤维素生物合成途径及其相关酶类还是知之甚少。近几年来,随着基因组学的发展,关于纤维素的生物合成及相关基因表达调控的研究也成绩斐然,现就高等植物纤维素生物合成途径及其相关的纤维素合成酶的最新研究进展作一介绍。     

Maturation by LctT Is Required for Biosynthesis of Full-Length Lantibiotic Lacticin 481

In lantibiotic lacticin 481 biosynthesis, LctT cleaves the precursor peptide and exports mature lantibiotic. Matrix-assisted laser desorption ionization-time of flight mass spectrometry revealed that a truncated form of lacticin 481 is produced in the absence of LctT or after cleavage site inactivation. Production of truncated lacticin 481 is 4-fold less efficient, and its specific activity is about 10-fold lower.     

Partial Purification and Some Properties of 7-Keto-8-aminopelargonic Acid Synthetase,an Enzyme Involved in Biotin Biosynthesis

Several genes that may be involved in embryogenesis have been isolated from somatic embryos of carrot by many workers. However, the function of these genes has not been discovered yet. As the first step toward finding the function of these genes, we established a rapid and efficient method for transformation of carrot by using direct embryogenesis from hypocotyl segments treated with 2,4-dichlorophenoxyacetic acid (2,4-D) for a short period.     

植物萜类生物合成中的后修饰酶

萜类化合物由于其结构类型丰富多样而被称为terpenome.除了参与植物生长发育、环境应答等生理过程,萜类化合物还应用于医药、有机化工等领域.萜类的生物合成大致可分为前体形成、骨架构建以及后修饰三部分,基本骨架通常由萜类合酶催化形成,进一步在后修饰酶的作用下产生数以万计的萜类化合物.结合我们对香茶菜二萜生物合成的初步研究结果,本文主要针对近年来植物萜类生物合成中的一些有代表性的后修饰酶包括P450单氧酶、双键还原酶、酰基转移酶和糖基转移酶,进行研究现状分析与展望.     

Partial Purification and Some Properties of 7-Keto-8-aminopelargonic Acid Synthetase,an Enzyme Involved in Biotin Biosynthesis

7-Keto-8-aminopelargonic acid synthetase (KAPA synthetase) which catalyzes the formation of KAPA from pimelyl CoA and l-alanine, and is involved in biotin biosynthesis, was partially purified from a cell-free extract of Bacillus sphaericus by a procedure involving ammonium sulfate fraction ation, protamine treatment, and DEAE-cellulose column chromatography. The reaction product was bioautographically confirmed to be KAPA. Some properties of the enzyme were also investigated. Among the amino acids, only l-alanine was active as a substrate, condensing with pimelyl CoA, The reaction required pyridoxal phosphate but the other vitamin B₆ compounds were inert. Typical inhibitors of pyridoxal phosphate enzymes showed marked inhibition to the reaction. Various amino acids such as l-cysteine, glycine, d-alanine, l-serine, l-histidine, and d-histidine were also found to be inhibitory.