限制和修饰系统LlaBIII在构建抗噬菌体菌株中的作用

限制和修饰 (ｒｅｓｔｒｉｃｔｉｏｎａｎｄｍｏｄｉｆｉｃａｔｉｏｎ ,Ｒ Ｍ)系统是指由限制性内切酶和甲基化酶组成的单亚基或多亚基复合酶系统 ,两者通常成对出现 ,具有相同的ＤＮＡ识别位点 ,其作用相反。Ｒ Ｍ系统在原核生物中普遍存在 ,在保护细胞免遭外源病毒侵害方面具有重要作用[1] 。作为发酵剂的乳酸乳球菌在乳制品发酵中具有重要作用 ,但这类菌株极易遭受噬菌体感染 ,导致菌株产酸力降低 ,甚至发酵失败 ,造成严重的经济损失。所以在乳制品发酵过程中防止噬菌体感染就成为十分重要的问题。通过自然筛选或诱变处理等手段筛选噬菌…     

限制和修饰系统LlaBⅢ在构建抗噬菌体菌株中的作用

限制和修饰(restriction and modification,R/M)系统是指由限制性内切酶和甲基化酶组成的单亚基或多亚基复合酶系统,两者通常成对出现,具有相同的DNA识别位点,其作用相反.R/M系统在原核生物中普遍存在,在保护细胞免遭外源病毒侵害方面具有重要作用[1].     

Potential of AbiS as defence mechanism determined by conductivity measurement

AIM: To compare pH and conductivity used in the determination of growth in reconstituted skim milk (RSM), to determine whether the presence of one or two plasmids in Lactococcus lactis had any influence on growth, and whether AbiS improved bacteriophages resistance of L. lactis. METHODS AND RESULTS: Conductivity and pH were used to determine growth in RSM. A small increase in the generation time was found with increasing number of plasmids, while their size was unimportant. The introduction of a plasmid-encoding AbiS did only enhance the level of phage resistance significant when other plasmids encoding either AbiS1 or the restriction modification system LlaBIII was present. CONCLUSIONS: The earliest detection of growth was observed by measuring pH, rather than conductance. The plasmid-encoded AbiS system has a potential to be used as a phage resistance mechanisms in L. lactis during milk fermentations, especially when combined with other anti-phage mechanisms. SIGNIFICANCE AND IMPACT OF THE STUDY: This study widened the knowledge about the influence of plasmid introduction on the growth rate of L. lactis, which is important for the construction of new strains. The level of protection against 936 groups of phages was only significant when the mechanism was present together with the RM system LlaBIII.     

Improvement of phage defence in Lactococcus lactis by introduction of the plasmid encoded restriction and modification system LlaAI

AIMS: To study the ability of the plasmid-encoded restriction and modification (R/M) system LlaAI to function as a bacteriophage resistance mechanism in Lactococcus lactis during milk fermentations. METHODS AND RESULTS: Plasmid pAIcat4, carrying the R/M system LlaAI and a chloramphenicol resistance cassette, was introduced into the plasmid-free strain L. lactis MG1614 and the industrial strain L. lactis 964. By measuring changes in conductivity the influence of different phage on the growth was determined. CONCLUSIONS: The plasmid-encoded R/M system LlaAI significantly improves the bacteriophage resistance of L. lactis during milk fermentations. SIGNIFICANCE AND IMPACT OF THE STUDY: It is essential to determine the potential of a phage defence mechanism in L. lactis starter culture strains during growth in milk before steps are taken to improve starter cultures. This study shows that LlaAI is useful for improvement of starter cultures.     

限制和修饰酶基因Lla BⅢ的克隆及分析

pJW566是从丹麦乳酪生产菌株Lactococcus lactis subsp.cremoris W56中分离到的,一个22.4kb,具有限制和修饰作用的质粒,内切酶ClaⅠ和pJW566不完全消化,所得片段与来自于质粒pVC5的氯霉素抗性基因连接得到一个携带有完整限制和修饰酶基因的质粒pJK1。基因亚克隆分析发现该基因位于约5kb的Sph0Ⅰ-Hin dⅢDNA片段上。序列分析表明该片段包含一个4572bp的开放阅读框架、编码一个由1576／1584个氨基酸残基组成的蛋白质,该基因命名为Lla BⅢ。蛋白质同源性查询发现在该蛋白的N－末端有7个保守区域,与R／M系统Ⅰ型和Ⅲ型内切酶有较高同源性,在蛋白的中间区域有4个代表N^6-腺苷酰甲基转移酶的特征序列,而蛋白的C－末端不同于任何已知蛋白。这种具有限制、修饰和可能的DNA识别作用的多功能蛋白,可能是一新的R／M系统。     

Cloning and analysis of the restriction-modification system LlaBI, a bacteriophage resistance system from Lactococcus lactis subsp. cremoris W56.

The genes coding for the type II restriction-modification (R/M) system LlaBI, which recognized the sequence 5'-C decreases TRYAG-3', have been cloned from a plasmid in Lactococcus lactis subsp. cremoris W56 and sequenced. The DNA sequence predicts an endonuclease of 299 amino acids (33 kDa) and a methylase of 580 amino acids (65 kDa). A 4.0-kb HindIII fragment in pSA3 was able to restrict bacteriophages, showing that the cloned R/M system can function as a phage defense mechanism in L. lactis.     

Identification of adsorption inhibition, restriction/modification and abortive infection type phage resistance systems in Lactococcus lactis strains

98 Lactococcus lactis strains were isolated from traditional fermented milk products in Turkey tested against 60 lactococcal lytic phages to determine their resistance levels. While 82 L. lactis strains were sensitive against lactic phages at different levels, 16 L. lactis strains showed resistance to all phages tested. Types of phage resistance among 16 L. lactis strains were identified as phage adsorption inhibition in eight strains, restriction/modification in six strains and abortive infection (heat sensitive phage resistance) in two strains, using three broad-spectrum phages phi pll 98-32, phi pld 67-42 and phi pld 67-44.     

Mobile CRISPR/Cas-Mediated Bacteriophage Resistance in Lactococcus lactis

Lactococcus lactis is a biotechnological workhorse for food fermentations and potentially therapeutic products and is therefore widely consumed by humans. It is predominantly used as a starter microbe for fermented dairy products, and specialized strains have adapted from a plant environment through reductive evolution and horizontal gene transfer as evidenced by the association of adventitious traits with mobile elements. Specifically, L. lactis has armed itself with a myriad of plasmid-encoded bacteriophage defensive systems to protect against viral predation. This known arsenal had not included CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins), which forms a remarkable microbial immunity system against invading DNA. Although CRISPR/Cas systems are common in the genomes of closely related lactic acid bacteria (LAB), none was identified within the eight published lactococcal genomes. Furthermore, a PCR-based search of the common LAB CRISPR/Cas systems (Types I and II) in 383 industrial L. lactis strains proved unsuccessful. Here we describe a novel, Type III, self-transmissible, plasmid-encoded, phage-interfering CRISPR/Cas discovered in L. lactis. The native CRISPR spacers confer resistance based on sequence identity to corresponding lactococcal phage. The interference is directed at phages problematic to the dairy industry, indicative of a responsive system. Moreover, targeting could be modified by engineering the spacer content. The 62.8-kb plasmid was shown to be conjugally transferrable to various strains. Its mobility should facilitate dissemination within microbial communities and provide a readily applicable system to naturally introduce CRISPR/Cas to industrially relevant strains for enhanced phage resistance and prevention against acquisition of undesirable genes.     

Evidence for a Plasmid-Linked Restriction-Modification System in Lactobacillus helveticus

The presence of a restriction-modification (R/M) system against two bacteriophages, 328-B1 and hv, was demonstrated in three Lactobacillus helveticus strains, CNRZ 1094, CNRZ 1095, and CNRZ 1096. In addition, the burst size of phage 328-B1 in the three restrictive strains CNRZ 1094, CNRZ 1095, and CNRZ 1096 was reduced with respect to the values obtained in its propagating strain, CNRZ 328. Heating at 60°C did not inactivate the R/M system. Nonrestrictive variants from CNRZ 1094 were easily obtained under several culture conditions, but treatment with novobiocin at 42°C followed by storage at −20°C resulted in drastic elimination of the R⁺/M⁺ phenotype from all clones tested. Electrophoretic analysis of CNRZ 1094 nonrestrictive variants revealed the concomitant loss of a 34-kb plasmid. Four EcoRI fragments from the 34-kb plasmid were cloned in the Escherichia coli vector pACYC184. The use of one or several of these fragments as probes confirmed the plasmidic location of the genes responsible for the R/M system. These probes also showed the presence of R/M plasmids in the two other restrictive strains, CNRZ 1095 and CNRZ 1096. Lactose-fermenting ability and/or proteolytic capacity was not linked to the 34-kb plasmid.     

Stacking of three different restriction and modification systems in Lactococcus lactis by cotransformation

Four plasmids encoding restriction and modification (R/M) systems are described that are different in the specificity of their restrictive activity toward the small isometric phage p2 and prolate phage c2. The R/M plasmids were cotransformed into Lactococcus lactis MG1363 with pVS2, encoding resistance to chloramphenicol and erythromycin, to indicate successful transformation events. Analysis of cotransformants showed that three different R/M plasmids could be combined in L. lactis MG1363. The efficiency at which phage plaqued on the transformants decreased as the number of R/M plasmids increased. Some plasmid combinations were unstable suggesting replicon incompatibility.     

Phenotypic and genetic characterization of the bacteriophage abortive infection mechanism AbiK from Lactococcus lactis.

The natural plasmid pSRQ800 isolated from Lactococcus lactis subsp. lactis W1 conferred strong phage resistance against small isometric phages of the 936 and P335 species when introduced into phage-sensitive L. lactis strains. It had very limited effect on prolate phages of the c2 species. The phage resistance mechanism encoded on pSRQ800 is a temperature-sensitive abortive infection system (Abi). Plasmid pSRQ800 was mapped, and the Abi genetic determinant was localized on a 4.5-kb EcoRI fragment. Cloning and sequencing of the 4.5-kb fragment allowed the identification of two large open reading frames. Deletion mutants showed that only orf1 was needed to produce the Abi phenotype. orf1 (renamed abiK) coded for a predicted protein of 599 amino acids (AbiK) with an estimated molecular size of 71.4 kDa and a pI of 7.98. DNA and protein sequence alignment programs found no significant homology with databases. However, a database query based on amino acid composition suggested that AbiK might be in the same protein family as AbiA. No phage DNA replication nor phage structural protein production was detected in infected AbiK+ L. lactis cells. This system is believed to act at or prior to phage DNA replication. WHen cloned into a high-copy vector, AbiK efficiency increased 100-fold. AbiK provides another powerful tool that can be useful in controlling phages during lactococcal fermentations.     

Additive Effects of Two Different Plasmid-Linked Restriction and Modification Systems in Lactococcus

Two plasmids, pND801 and pND802, encoding different restriction and modification systems were isolated from Lactococcus lactis ssp. lactis LL42-1 and Lactococcus lactis ssp. cremoris LC14-1, respectively. pND802 contained one Sphl restriction enzyme site and the whole plasmid was cloned into the Sphl site of the streptococcal/ E. coli shuttle vector pSA3 generating the plasmid pND803. pND803 was stably maintained in L.lactis MG1363 harbouring pND801. The combination of the two R/M systems within L.lactis MG1363 resulted in an additive resistance towards both isometric phage and prolate phage.     

A Starter Culture Rotation Strategy Incorporating Paired Restriction/ Modification and Abortive Infection Bacteriophage Defenses in a Single Lactococcus lactis Strain

Three derivatives of Lactococcus lactis subsp. lactis NCK203, each with a different pair of restriction/ modification (R/M) and abortive infection (Abi) phage defense systems, were constructed and then rotated in repeated cycles of a milk starter culture activity test (SAT). The rotation proceeded successfully through nine successive SATs in the presence of phage and whey containing phage from previous cycles. Lactococcus cultures were challenged with 2 small isometric-headed phages, (phi)31 and ul36, in one rotation series and with a composite of 10 industrial phages in another series. Two native lactococcal R(sup+)/M(sup+) plasmids, pTRK68 and pTRK11, and one recombinant plasmid, pTRK308, harboring a third distinct R/M system were incorporated into three NCK203 derivatives constructed separately for the rotation. The R(sup+)/M(sup+) NCK203 derivatives were transformed with high-copy-number plasmids encoding four Abi genes, abiA, abiC, per31, and per50. Various Abi and R/M combinations constructed in NCK203 were evaluated for their effects on cell growth, level of phage resistance, and retardation of phage development during repeated cycles of the SAT. The three NCK203 derivatives chosen for use in the SAT exhibited additive effects of the R/M and Abi phenotypes against sensitive phages. In such combinations, phage escaping restriction are prevented from completing their infective cycle by an abortive response that kills the host cell. The rotation series successfully controlled modified, recombinant, and mutant phages which were resistant to any one of the individual defense systems by presenting a different set of R/M and Abi defenses in the next test of the rotation.     

Molecular Properties of Streptococcus thermophilus Plasmid pER35 Encoding a Restriction Modification System

Bacteriophage attack on lactic fermentation bacteria (LFB) is costly to the dairy industry because it results in product loss. One mechanism used by LFB to protect themselves from bacteriophage attack is restriction of foreign DNA. Three plasmids, pER16, pER35, and pER36, from three different strains of the thermotolerant dairy fermentation bacterium Streptococcus thermophilus were sequenced. One of these plasmids, pER35, isolated from S. thermophilus ST135, encoded a type IC restriction-modification (R-M) system very similar to those encoded on plasmids pIL2614 in Lactococcus lactis subsp. lactis and pND861 in Lactococcus lactis biovar diacetylactis. The high degree of identity between the R-M systems encoded on pER35, pIL2614, and pND861 indicated the potential for horizontal transfer of these genes between different species of lactic fermentation bacteria. Similar to the functional R-M system encoded on pIL2614 that protects the mesophilic L. lactis subsp. lactis against phage attack, the R-M system on pER35 most likely functions in the same role in S. thermophilus ST135. The plasmid pER16 was found to encode the specificity subunit of the R-M system, but not the R or M subunits. In addition, all three plasmids encoded proteins that are present on other S. thermophilus plasmids, including a protein for rolling-circle replication (RepA) and a low-molecular-weight stress protein (Hsp). The presence of a complete R-M system encoded on a plasmid in S. thermophilus, a species that often lacks plasmids, is novel and may be beneficial for protecting S. thermophilus from bacteriophage attack under dairy fermentation conditions.     

Cloning and sequencing of LlaDCHI [corrected] restriction/modification genes from Lactococcus lactis and relatedness of this system to the Streptococcus pneumoniae DpnII system.

The natural 7.8-kb plasmid pSRQ700 was isolated from Lactococcus lactis subsp. cremoris DCH-4. It encodes a restriction/modification system named LlaDCHI [corrected]. When introduced into a phage-sensitive L. lactis strain, pSRQ700 confers strong phage resistance against the three most common lactococcal phage species, namely, 936, c2, and P335. The LlaDCHI [corrected] endonuclease was purified and found to cleave the palindromic sequence 5'-GATC-3'. It is an isoschizomer of Streptococcus pneumoniae DpnII. The plasmid pSRQ700 was mapped, and the genetic organization of LlaDCHI [corrected] was localized. Cloning and sequencing of the entire LlaDCHI [corrected] system allowed the identification of three open reading frames. The three genes (llaIIA, llaIIB, and llaIIC) overlapped and are under one putative promoter. A putative terminator was found at the end of llaIIC. The genes llaIIA and llaIIB coded for m6A methyltransferases, and llaIIC coded for an endonuclease. The LlaDCHI [corrected] system shares strong genetic similarities with the DpnII system. The deduced amino acid sequence of M.LlaIIA was 75% identical with that of M.DpnII, whereas M.LlaIIB was 88% identical with M.DpnA. However, R.LlalII shared only 31% identity with R.DpnII.     

乳酸乳球菌乳脂亚种W56中质粒pJW566的生物学特性

从丹麦乳酪发酵启子乳酸乳球菌乳脂亚种 (Lactococcuslactissubsp .cremoris)W56中 ,分离到一个 2 2 4kb的质粒pJW566,将该质粒转化到无质粒且噬菌体敏感的L .lactisMG1 61 4、SMQ86菌株中 ,所得转化子对常见 963、c2和P335属的噬菌体具有一定抗性。经测定噬菌体以及含有pJW566的菌株所繁育的噬菌体效价 ,发现该质粒对外源DNA具有限制和修饰 (Re strictionandModification ,R M)作用。将pJW566转化到一株噬菌体敏感的乳酪工业生产菌株L .lactisCHCC2 2 81 ,在牛奶发酵中 ,表现出较强的噬菌体抗性。体外内切酶活性测定表明 ,该质粒具有的限制性内切酶需要Mg2 +和ATP ,而AdoMet(S adenosylmethionine,AdoMet)对酶活有促进作用     

Characterization of the two-component abortive phage infection mechanism AbiT from Lactococcus lactis

During the production of fermented dairy products, virulent bacteriophages infecting Lactococcus lactis can delay or stop the milk acidification process. A solution to this biological problem consists of introducing natural phage barriers into the strains used by the dairy industry. One such hurdle is called abortive infection (Abi) and causes premature cell death with no or little phage progeny. Here, we describe the isolation and characterization of a novel Abi mechanism encoded by plasmid pED1 from L. lactis. The system is composed of two constitutively cotranscribed genes encoding putative proteins of 127 and 213 amino acids, named AbiTi and AbiTii, respectively. Site-directed mutagenesis indicated that a hydrophobic region at the C-terminal extremity of AbiTi is essential to the antiphage phenotype. The AbiT system is effective against phages of the 936 and P335 species (efficiency of plaquing between 10(-5) and 10(-7)) and causes a 20-fold reduction in the efficiency to form centers of infection as well as a 10- to 12-fold reduction in the burst size. Its efficacy could be improved by raising the plasmid copy number, but changing the intrinsic ratio of AbiTi and AbiTii did not greatly affect the antiphage activity. The monitoring of the intracellular phage infection process by DNA replication, gene expression, and electron microscopy as well as the study of phage mutants by genome mapping indicated that AbiT is likely to act at a later stage of the phage lytic cycle.     

Development of food-grade cloning and expression vectors for Lactococcus lactis

AIMS: To develop food-grade cloning and expression vectors for use in genetic modification of Lactococcus lactis. METHODS AND RESULTS: Two plasmid replicons and three dominant selection markers were isolated from L. lactis and used to construct five food-grade cloning vectors. These vectors were composed of DNA only from L. lactis and contained no antibiotic resistance markers. Three of the vectors (pND632, pND648 and pND969) were based on the same plasmid replicon and carried, either alone or in combination, the three different selectable markers encoding resistance to nisin, cadmium and/or copper. The other two (pND965DJ and pND965RS) were derived from a cadmium resistance plasmid, and carried a constitutive promoter and a copper-inducible promoter, respectively, immediately upstream of a multicloning site. All vectors were stable in L. lactis LM0230 for at least 40 generations without selection pressure. The two groups of vectors were compatible in L. lactis LM0230. The vectors pND648 and pND965RS, as representatives of the two groups, were transferred successfully by electroporation into and maintained in an industrial strain of L. lactis. The usefulness of the vectors was further demonstrated by expressing a phage resistance gene (abiI) in another industrial strain of L. lactis. CONCLUSIONS: The five food-grade vectors constructed are potentially useful for industrial strains of L. lactis. SIGNIFICANCE AND IMPACT OF THE STUDY: These vectors represent a new set of molecular tools useful for food-grade modifications of L. lactis.     

Isolation and characterization of large lactococcal phage resistance plasmids by pulsed-field gel electrophoresis

Abstract Five phage-resistant Lactococcus lactis strains were able to transfer by conjugation the lactose-fermenting ability (Lac⁺) to a plasmid-free Lac⁻ L. lactis strain. In each case, some Lac⁺ transconjugants were phage-resistant and contained one or two additional plasmids of high molecular mass, as demonstrated by pulsed-field gel electrophoresis. Plasmids pPF144 (144 kb), pPF107 (107 kb), pPF118 (118 kb), pPF72 (72 kb) and pPF66 (66 kb) were characterized: they are conjugative (Tra⁺), they confer a phage-resistant phenotype and they bear lactose-fermenting ability (Lactose plasmid) except for the last two. Plasmids pPF144, pPF107 and pPF118 resulted probably from a cointegrate formation between the Lactose plasmid and another plasmid of the donor strain, whereas pPF72, pPF66 and the Lactose plasmid were distinct in the corresponding transconjugants. Plasmids pPF72 and pPF66 produced a bacteriocin. At 30°C, the phage resistance conferred by the plasmids was complete against small isometric-headed phage and partial against prolate-headed phage, except for pPF107 whose phage resistance mechanism was totally effective against both types of phages, but was completely inactivated at 40°C. Restriction maps of four of the plasmids were constructed using pulsed-field gel electrophoresis.