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ABSTRACT: Survival is assuredly the prime directive for all living organisms either as individuals or as a species. One of the main challenges encountered by bacterial populations is the danger of bacteriophage attacks, since infection of a single bacterium may rapidly propagate, decimating the entire population. In order to protect themselves against this acute threat, bacteria have developed an array of defence mechanisms, which range from preventing the infection itself via interference with bacteriophage adsorption to the cell surface and prevention of phage DNA injection, to degradation of the injected phage DNA. This last defence mechanism is catalysed by the bacterial restriction-modification (R-M) systems, and in particular, by nucleoside 5'-triphosphate (NTP)-dependent restriction enzymes, e.g. type I and type III R-M systems or the modification-dependent endonucleases. Type I and type III restriction systems have dual properties. They may either act as methylases and protect the host's own DNA against restriction by methylating specific residues, or they catalyse ATP-dependent endonuclease activity so that invading foreign DNA lacking the host-specific methylation is degraded. These defence mechanism systems are further complemented by the presence of methylation-dependent, GTP-dependent endonucleases, that restricts specifically methylated DNA. Although all three types of endonucleases are structurally very different, they share a common functional mechanism. They recognise and bind to specific DNA sequences but do not cleave DNA within those target sites. They belong to the general class of DNA motor proteins, which use the free energy associated with nucleoside 5'-triphosphate hydrolysis to translocate DNA so that the subsequent DNA cleavage event occurs at a distance from the endonuclease recognition site. Moreover, DNA cleavage appears to be a random process triggered upon stalling of the DNA translocation process and requiring dimerisation of the bound endonucleases for a concerted break of both DNA strands. In this review, we present a detailed description and analysis of the functional mechanism of the three known NTP-dependent restriction systems: type I and type III restriction-modification enzymes, as well as the methylation-dependent McrBC endonuclease.
Biochimie 12/2002; 84(11):1047-59. · 3.02 Impact Factor
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ABSTRACT: The plasmids pN42 and pJBL2 were isolated from the Lactobacillus delbrueckii subsp. lactis strains NCC88 and JCL414. DNA sequence determination and bioinformatic analysis revealed a strikingly conserved genetic organization containing five major, highly conserved open reading frames (ORFs). Transformation studies indicated that ORF2 (consisting of a primase fused to a replicative DNA helicase), ori, and ORF3 constitute the minimal requirements for replication of pN42 in the heterologous host Lactococcus lactis. The ORF1's are predicted to encode type I restriction-modification (R-M) system HsdS subunits with different specificities on either plasmid, suggesting that these plasmids may be involved in host defense by expanding their host R-M system repertoire. These plasmids constitute the basis for the construction of novel L. delbrueckii vectors.
Plasmid 04/2002; 47(2):153-7. · 1.52 Impact Factor
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ABSTRACT: Lactic acid bacteria have evolved in a very rich and specific medium, milk, and have in the course of adaptation discarded non-essential metabolic processes. Thus, Lactobacillus delbrueckii subsp. lactis lacking both the citric cycle and the pentose-phosphate pathway relies mainly on glycolysis for ATP production and in particular, the reaction catalyzed by pyruvate kinase (PYK). In this study, we purified and characterized L. delbrueckii lactis PYK and studied its role in metabolic regulation. L. delbrueckii lactis (PYK) is a classical homotetramer and a V-type allosteric enzyme. Positive effectors are fructose 1,6-bisphosphate (Ka=2.2 mm), as well as fructose 6-phosphate and glucose 6-phosphate, which were found to be 1.3- and 1.6-fold more efficient activators than fructose 1,6-bisphosphate, respectively. L. delbrueckii lactis PYK is inhibited by inorganic phosphate, high concentrations of phosphoenolpyruvate and ATP (EC50=0.75 mm). An ATP-binding motif identified on the 134-amino acid C-terminal extension of the enzyme might be involved in ATP regulation of L. delbrueckii lactis PYK. This extension also encodes a protein domain with homologies to the enzyme I of the Escherichia coli sugar-PTS system suggesting that the PYK might be involved in L. delbrueckii lactis sugar transport metabolism.
International Dairy Journal.
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ABSTRACT: The plasmids pN42 and pJBL2 were isolated from the Lactobacillus delbrueckii subsp. lactis strains NCC88 and JCL414. DNA sequence determination and bioinformatic analysis revealed a strikingly conserved genetic organization containing five major, highly conserved open reading frames (ORFs). Transformation studies indicated that ORF2 (consisting of a primase fused to a replicative DNA helicase), ori, and ORF3 constitute the minimal requirements for replication of pN42 in the heterologous host Lactococcus lactis. The ORF1's are predicted to encode type I restriction-modification (R-M) system HsdS subunits with different specificities on either plasmid, suggesting that these plasmids may be involved in host defense by expanding their host R-M system repertoire. These plasmids constitute the basis for the construction of novel L. delbrueckii vectors.
Plasmid.
