Nucleolytic activities in Lactococcus lactis subsp. lactis NCDO 497

Mayo, Baltasar

doi:10.1016/0378-1097(91)90085-o

Cited by 10 publications

(12 citation statements)

References 4 publications

(5 reference statements)

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“…In contrast, serF/BM appeared to be dispensable with respect to maintenace of the ENase gene. and the renamed Lla497I (formerly LlaI; Mayo et al 1991) with the recognition sequences 5'-CC/NGG-3' and 5'-CC/wGG-3', respectively. The ENase gene, serF/R, displayed a unique amino acid sequence even though it is an isoschizomer of SsoILR.…”

Section: Type II Rim Systemsmentioning

confidence: 99%

Bacteriophage defence systems in lactic acid bacteria

Forde¹,

Fitzgerald²

1999

Lactic Acid Bacteria: Genetics, Metabolism and Applications

106

131

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The study of the interactions between lactic acid bacteria and their bacteriophages has been a vibrant and rewarding research activity for a considerable number of years. In the more recent past, the application of molecular genetics for the analysis of phage-host relationships has contributed enormously to the unravelling of specific events which dictate insensitivity to bacteriophage infection and has revealed that while they are complex and intricate in nature, they are also extremely effective. In addition, the strategy has laid solid foundations for the construction of phage resistant strains for use in commercial applications and has provided a sound basis for continued investigations into existing, naturally-derived and novel, genetically-engineered defence systems. Of course, it has also become clear that phage particles are highly dynamic in their response to those defence systems which they do encounter and that they can readily adapt to them as a consequence of their genetic flexibility and plasticity. This paper reviews the exciting developments that have been described in the literature regarding the study of phage-host interactions in lactic acid bacteria and the innovative approaches that can be taken to exploit this basic information for curtailing phage infection.

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Section: Type II Rim Systemsmentioning

confidence: 99%

Bacteriophage defence systems in lactic acid bacteria

Forde¹,

Fitzgerald²

1999

Lactic Acid Bacteria: Genetics, Metabolism and Applications

106

131

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“…It should be noted that the original LlaI designation for the pTR2030-encoded restriction enzyme was proposed by Hill et al (18) following sequence characterization of the methylase gene. However, in a separate and unrelated instance, the LlaI designation was subsequently used to describe an observed type II endonuclease activity in another lactococcal strain (27). As numerous subsequent citations have referred to LlaI as the one proposed by Hill et al (18), we will continue to do likewise.…”

mentioning

confidence: 93%

In vivo restriction by LlaI is encoded by three genes, arranged in an operon with llaIM, on the conjugative Lactococcus plasmid pTR2030

1995

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The LlaI restriction and modification (R/M) system is encoded on pTR2030, a 46.2-kb conjugative plasmid from Lactococcus lactis. The llaI methylase gene, sequenced previously, encodes a functional type IIS methylase and is located ϳ5 kb upstream from the abiA gene, encoding abortive phage resistance. In this study, the sequence of the region between llaIM and abiA was determined and revealed four consecutive open reading frames (ORFs). Northern (RNA) analysis showed that the four ORFs were part of a 7-kb operon with llaIM and the downstream abiA gene on a separate transcriptional unit. The deduced protein sequence of ORF2 revealed a P-loop consensus motif for ATP/GTP-binding sites and a three-part consensus motif for GTP-binding proteins. Data bank searches with the deduced protein sequences for all four ORFs revealed no homology except for ORF2 with McrB, in three regions that coincided with the GTP-binding motifs in both proteins. To phenotypically analyze the llaI operon, a 9.0-kb fragment was cloned into a high-copy-number lactococcal shuttle vector, pTRKH2. The resulting construct, pTRK370, exhibited a significantly higher level of in vivo restriction and modification in L. lactis NCK203 than the low-copy-number parental plasmid, pTR2030. A combination of deletion constructions and frameshift mutations indicated that the first three ORFs were involved in LlaI restriction, and they were therefore designated llaI.1, llaI.2, and llaI.3. Mutating llaI.1 completely abolished restriction, while disrupting llaI.2 or llaI.3 allowed an inefficient restriction of phage DNA to occur, manifested primarily by a variable plaque phenotype. ORF4 had no discernible effect on in vivo restriction. A frameshift mutation in llaIM proved lethal to L. lactis NCK203, implying that the restriction component was active without the modification subunit. These results suggested that the LlaI R/M system is unlike any other R/M system studied to date and has diverged from the type IIS class of restriction enzymes by acquiring some characteristics reminiscent of type I enzymes.The biological role of restriction and modification (R/M) systems is to protect cells from invasion by foreign DNA, such as promiscuous plasmids or infecting bacteriophages. The restriction enzyme can discriminate between foreign and host DNA by the presence of methylation on certain nucleotides (reviewed in reference 4). Traditionally, these enzymes have been classified into three types. Type I enzymes consist of three subunits which are responsible for modification, restriction, and specificity. A complex of the three subunits is required to restrict DNA in the presence of Mg 2ϩ , ATP, and S-adenosylmethionine. The site of cleavage is generally a large distance (often Ͼ1 kb) from the recognition site. Type II enzymes are the most common and the simplest, with over 1,000 different varieties identified (45). They consist of one subunit, require Mg 2ϩ as a cofactor, and restrict at or near their recognition sites. Those that restrict outside their recognition sites ...

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“…The existence of most R/M systems in lactococci has been deduced from the results of phage restriction studies. However, four systems have been characterized by enzyme purification and characterization of the DNA target (13,22,26,27) or by cloning and sequencing of the corresponding genes (10, 24, 27-29, 42, 43). Three of these systems are type II systems, and one (29), comprising three genes associated with restriction activity and a type IIs methylase, has not been classified.…”

Section: Discussionmentioning

confidence: 99%

Lla FI, a Type III Restriction and Modification System in Lactococcus lactis

Su¹,

Hsieh

et al. 1999

Appl Environ Microbiol

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We describe a type III restriction and modification (R/M) system,LlaFI, in Lactococcus lactis. LlaFI is encoded by a 12-kb native plasmid, pND801, harbored in L. lactisLL42-1. Sequencing revealed two adjacent open reading frames (ORFs). One ORF encodes a 680-amino-acid polypeptide, and this ORF is followed by a second ORF which encodes an 873-amino-acid polypeptide. The two ORFs appear to be organized in an operon. A homology search revealed that the two ORFs exhibited significant similarity to type III restriction (Res) and modification (Mod) subunits. The complete amino acid sequence of the Mod subunit of LlaFI was aligned with the amino acid sequences of four previously described type III methyltransferases. Both the N-terminal regions and the C-terminal regions of the Mod proteins are conserved, while the central regions are more variable. An S-adenosyl methionine (Ado-Met) binding motif (present in all adenine methyltransferases) was found in the N-terminal region of the Mod protein. The seven conserved helicase motifs found in the previously described type III R/M systems were found at the same relative positions in the LlaFI Res sequence.LlaFI has cofactor requirements for activity that are characteristic of the previously described type III enzymes. ATP and Mg2+ are required for endonucleolytic activity; however, the activity is not strictly dependent on the presence of Ado-Met but is stimulated by it. To our knowledge, this is the first type III R/M system that has been characterized not just in lactic acid bacteria but also in gram-positive bacteria.

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Nucleolytic activities in Lactococcus lactis subsp. lactis NCDO 497

Cited by 10 publications

References 4 publications

Bacteriophage defence systems in lactic acid bacteria

Bacteriophage defence systems in lactic acid bacteria

In vivo restriction by LlaI is encoded by three genes, arranged in an operon with llaIM, on the conjugative Lactococcus plasmid pTR2030

Lla FI, a Type III Restriction and Modification System in Lactococcus lactis

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