Publisher Summary This chapter discusses the interaction of restriction endonucleases with double-stranded DNA molecules at specific sites leading to cleavage of the DNA into a number of fragments. The specificity of this interaction is thought to depend on the recognition by the enzyme of a particular sequence of base-pairs on the substrate DNA. Restriction endonucleases are found in many bacterial strains as products of genes carried either on the bacterial chromosome or on plasmid DNA. Among enzymes obtained from independent sources, each usually shows its own specificity of interaction. The chapter describes that a bacterial strain can protect its own DNA from cleavage by its restriction endonucleases. This protection is brought about by site-specific methylation of the DNA, for which another activity, the DNA modification methylase, is responsible. Both endonuclease and methylase are thought to recognize the same base sequences on their substrate DNA. Each independent system of restriction and modification activities would then recognize its own particular target on the DNA. Therefore, the modification given by a particular methylase protects the DNA only against restriction by the correlated endonuclease. The chapter also discusses that mostly for reasons of a historical and practical nature, laboratory strains such as Escherichia coli K12 and B or Haemophilus influenzae are widely used in the experimental investigation of DNA restriction and modification, although many other bacteria are either known or are likely to have restriction and modification systems.

DNA Modification and Restriction

A Suggested nomenclature for bacterial host modification and restriction systems and their enzymes

SummaryE. coli 15T- carries two distinct sets of DNA restriction and modification activities. The genetic information for system A is contained in the bacterial chromosome and linked to the thr region. This fact suggests host specificity A to be related to those of strains K and B. The genes controlling system 15 are on a plasmid which is related to phage Pl: it competes with Pl for stable inheritance in the carried state and it genetically recombines with Pl. This recombination may produce plasmid genomes with newly assorted characters (see Table 3). One of them is an active, Pl-like prophage with the 15-specific instead of the parental Pl-specific restriction and modification characters. Superinfection of 15T- with Pl may also result in curing of the bacteria from the restriction plasmid.Bot A- and 15-specific restrictions and modifications act on bacterial DNA, on the DNA of various sex factors and on the DNA of certain bacteriophages, e.g. of phage λ. Phage 82 DNA is sensitive only to 15-specific restriction, but not to A-specific restriction.Independently of the A- and 15-specific restrictions, the growth of phage λ in E. coli 15T- encounters another limitation of yet unknown nature. No such limitation is observed either with phage 82 or with mutants of λ occurring at a frequency of about 10-5.

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Host specificity of DNA produced by Escherichia coli. XII. The two restriction and modification systems of strain 15T

The auto-induction method of protein expression in E. coli is based on diauxic growth resulting from dynamic function of lac operon regulatory elements (lacO and LacI) in mixtures of glucose, glycerol, and lactose. The results show that successful execution of auto-induction is strongly dependent on the plasmid promoter and repressor construction, on the oxygenation state of the culture, and on the composition of the auto-induction medium. Thus expression hosts expressing high levels of LacI during aerobic growth exhibit reduced ability to effectively complete the auto-induction process. Manipulation of the promoter to decrease the expression of LacI altered the preference for lactose consumption in a manner that led to increased protein expression and partially relieved the sensitivity of the auto-induction process to the oxygenation state of the culture. Factorial design methods were used to optimize the chemically defined growth medium used for expression of two model proteins, Photinus luciferase and enhanced green fluorescent protein, including variations for production of both unlabeled and selenomethionine-labeled samples. The optimization included studies of the expression from T7 and T7-lacI promoter plasmids and from T5 phage promoter plasmids expressing two levels of LacI. Upon the basis of the analysis of over 500 independent expression results, combinations of optimized expression media and expression plasmids that gave protein yields of greater than 1000 mug/mL of expression culture were identified.

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Enhanced bacterial protein expression during auto-induction obtained by alteration of lac repressor dosage and medium composition.

Naturally occurring modification of the canonical A, G, C, and T bases can be found in the DNA of cellular organisms and viruses from all domains of life. Bacterial viruses (bacteriophages) are a particularly rich but still underexploited source of such modified variant nucleotides. The modifications conserve the coding and base-pairing functions of DNA, but add regulatory and protective functions. In prokaryotes, modified bases appear primarily to be part of an arms race between bacteriophages (and other genomic parasites) and their hosts, although, as in eukaryotes, some modifications have been adapted to convey epigenetic information. The first half of this review catalogs the identification and diversity of DNA modifications found in bacteria and bacteriophages. What is known about the biogenesis, context, and function of these modifications are also described. The second part of the review places these DNA modifications in the context of the arms race between bacteria and bacteriophages. It focuses p...

http://pubs.acs.org/doi/pdf/10.1021/acs.chemrev.6b00114

Biosynthesis and Function of Modified Bases in Bacteria and Their Viruses

Bacteria with A-specific restriction plate unmodified phage λ with an efficiency of 10-2. One mutational event can produce restriction insensitive (sAo) mutants of λ. These differ from the original sA form of λ by no other property than their response to A-host specificity. Two-parental phage crosses involving sA and sAo, respectively, as non-selective marker allowed to map sA between genes cII and O. These data indicate that sA is the only site on λDNA with affinity for A-specific restriction. λDNA is thus an interesting substrate in in vitro A-specific restriction and modification. Using an assay based on the infectivity of λDNA on helper-infected bacteria, A-specific modification activity was found in partially purified sonicates of bacteria with A-host specificity. In parallel to modification, 3H-methyl label from s-adenosylmethionine, the only cofactor required for modification, was transferred to unmodified λDNA. No association of radioactivity was observed in control experiments with DNA from either modified λ·A or from aλsAo mutant. These data suggest that A-specific modification is brought about by DNA methylation and that the sAo mutation not only abolished the affinity for A-specific restriction, but also for A-specific modification.

Host specificity of DNA produced by Escherichia coli. XVI. Phage lambda DNA carries a single site of affinity for A-specific restriction and modification.

Alain Rifat

Papers

Host specificity of DNA produced by Escherichia coli. XVI. Phage lambda DNA carries a single site of affinity for A-specific restriction and modification.