Evolutionary relationship of Alw26I, Eco31I and Esp3I, restriction endonucleases that recognise overlapping sequences.

Type II restriction endonucleases (ENases) have served as models for understanding the enzyme-based site-specific cleavage of DNA. Using the knowledge gained from the available crystal structures, a number of attempts have been made to alter the specificity of ENases by mutagenesis. The negative results of these experiments argue that the three-dimensional structure of DNA-ENase complexes does not provide enough information to enable us to understand the interactions between DNA and ENases in detail. This conclusion calls for alternative approaches to the study of structure-function relationships related to the specificity of ENases. Comparative analysis of ENases that manifest divergent substrate specificities, but at the same time are evolutionarily related to each other, may be helpful in this respect. The success of such studies depends to a great extent on the availability of related ENases that recognise partially overlapping nucleotide sequences (e.g. sets of enzymes that bind to recognition sites of increasing length). In this study we report the cloning and sequence analysis of genes for three Type IIS restriction-modification (RM) systems. The genes encoding the ENases Alw26I, Eco31I and Esp3I (whose recognition sequences are 5'-GTCTC-3', 5'-GGTCTC-3' and 5'-CGTCTC-3', respectively) and their accompanying methyltransferases (MTases) have been cloned and the deduced amino acid sequences of their products have been compared. In pairwise comparisons, the degree of sequence identity between Alw26I, Eco31I and Esp3I ENases is higher than that observed hitherto among ENases that recognise partially overlapping nucleotide sequences. The sequences of Alw26I, Eco31I and Esp3I also reveal identical mosaic patterns of sequence conservation, which supports the idea that they are evolutionarily related and suggests that they should show a high level of structural similarity. Thus these ENases represent very attractive models for the study of the molecular basis of variation in the specific recognition of DNA targets. The corresponding MTases are represented by proteins of unusual structural and functional organisation. Both M. Alw26I and M. Esp3I are represented by a single bifunctional protein, which is composed of an m(6)A-MTase domain fused to a m(5)C-MTase domain. In contrast, two separate genes encode the m(6)A-MTase and m(5)C-MTase in the Eco31I RM system. Among the known bacterial m(5)C-MTases, the m(5)C-MTases of M. Alw26I, M. Eco31I and M. Esp3I represent unique examples of the circular permutation of their putative target recognition domains together with the conserved motifs IX and X.

Discovery and distribution of super-integrons among pseudomonads.

Until recently, integrons (systems for acquisition and expression of new genetic materials) have been associated generally with antibiotic resistance gene cassettes. The discovery of 'super-integrons' in Vibrionaceae suggests a greater impact of this gene acquisition mechanism on bacterial genome evolution than initially believed. Super-integrons may contain more than 100 gene cassettes and may encode other determinants, including biochemical functions or virulence factors. Here, we report the genetic organization of a super-integron from Pseudomonas alcaligenes ATCC 55044. This is the first evidence of a super-integron in a non-pathogenic bacterium, one which is widely distributed in a great number of ecological niches such as soil and aquatic habitats. Here, the sequence composition, open reading frame (ORF) content and organization of In55044 are described and found to have features intermediate between the multidrug-resistant integrons and the Vibrio cholerae super-integron. Similar structures are inferred to be present in several Pseudomonas species, based on polymerase chain reaction (PCR) experiments.

The LipB protein is a negative regulator of dam gene expression in Escherichia coli.

Transcription initiation of the major promoter (P2) of the Escherichia coli dam gene increases with growth rate. The presence of three partially palindromic motifs, (TTCAGT(N(20))TGAG), designated G (growth)-boxes, within the -52 to +31 region of the promoter, may be related to growth rate control. Deletion of two of these repeats, downstream of the transcription initiation point, result in constitutive high activity of the promoter. The unlinked cde-4::miniTn10 insertion also results in severalfold higher activity of the dam P2 promoter, suggesting that this mutation resulted in the loss of a putative dam P2 repressor. The cde-4 mutation was mapped to the lipB (lipoic acid) gene, which we show encodes a 24 kDa protein initiating at a TTG codon. LipB is a highly conserved protein in animal and plant species, other bacteria, Archaea, and yeast. Plasmids expressing the native or hexahistidine-tagged LipB complement the phenotype of the cde-4 mutant strain. The level of LipB in vivo was higher in exponentially growing cells than those in the stationary phase. Three G-box motifs were also found in the lipB region. Models for the regulation of expression of the two genes are discussed.

The eco72IC gene specifies a trans-acting factor which influences expression of both DNA methyltransferase and endonuclease from the Eco72I restriction-modification system.

Eco72I from Escherichia coli RFL72 is a type-II restriction-modification (R-M) system recognizing and cleaving the sequence 5'-CAC decreases GTG-3'. The R-M genes are transcribed divergently and between the two genes is a small open reading frame codirectional to the R gene. This small ORF acts both to stimulate ENase expression and to depress DNA methyltransferase synthesis. The activity of beta Gal produced from the eco72IM::lacZ translational fusion increased tenfold, and eco72IR::lacZ translational fusion beta Gal activity decreased 130-fold when eco72IC was inactivated by a frameshift mutation. Analysis of nucleotide sequences of R-M systems, containing C genes, revealed a 5'-ACCTTATAGTC-3' consensus sequence upstream from the regulatory genes in all six analysed R-M systems. This sequence, named C-box, may play the role of an operator sequence.

Cloning of the ppu21IM gene using a in vivo selection method.

A genetic system enabling the in vivo selection of genes encoding the DNA-modifying enzymes was developed. A gene library is transformed into a strain harboring the restriction-modification (R-M) system which a recognition sequence is a subset of the target sequence of the DNA methyltransferase (MTase) to be cloned. If the residing MTase is temperature sensitive, the inability of transformants to grow at 42 degrees C provides a simple and convenient procedure for the isolation of new MTase-encoding genes. The feasibility of this procedure has been demonstrated by the isolation of the ppu21IM gene from a Pseudomonas putida RFL21 gene library.

Identification of a gene encoding a DNA invertase-like enzyme adjacent to the PaeR7I restriction-modification system.

A gene encoding a DNA invertase-like enzyme was identified adjacent to the PaeR7I restriction-modification system (R-M), and was named paeR7IN (N for iNvertase). Sequence analysis revealed that this gene has the same polarity as the PaeR7IRM operon, and would encode a polypeptide of 21,506 Da. An amino-acid sequence similarity of 45-49% was found between the deduced protein product and various DNA invertases.

CAATTG-specific restriction-modification munI genes from Mycoplasma: sequence similarities between R.MunI and R.EcoRI.

The genes coding for the MunI restriction-modification (R-M) system, which recognize the sequence 5'-CAATTG, have been cloned and expressed in Escherichia coli, and their nucleotide sequences have been determined. The restriction endonuclease (ENase; R.MunI) is encoded by an open reading frame (ORF) of 606 bp, and a 699-bp ORF codes for the methyltransferase (MTase). The two genes are transcribed divergently from a 355-bp region. The gene encoding the ENase is preceded by a short co-linear ORF of 222 bp. The deduced amino acid (aa) sequence of this short ORF (SORF) closely resembles the sequences of a family of regulatory proteins that are associated with other type-II R-M systems. Comparative analysis of the deduced aa sequence of R.MunI revealed several regions of similarity to the EcoRI and RsrI ENases that recognize the GAATTC sequence. The similar mode of interaction of MunI, EcoRI and RsrI with the tetranucleotide AATT, common to the recognition sequences of these ENases, was suggested.

Cloning and sequence analysis of the genes coding for Eco57I type IV restriction-modification enzymes.

A 6.3 kb fragment of E.coli RFL57 DNA coding for the type IV restriction-modification system Eco57I was cloned and expressed in E.coli RR1. A 5775 bp region of the cloned fragment was sequenced which contains three open reading frames (ORF). The methylase gene is 1623 bp long, corresponding to a protein of 543 amino acids (62 kDa); the endonuclease gene is 2991 bp in length (997 amino acids, 117 kDa). The two genes are transcribed convergently from different strands with their 3'-ends separated by 69 bp. The third short open reading frame (186 bp, 62 amino acids) has been identified, that precedes and overlaps by 7 nucleotides the ORF encoding the methylase. Comparison of the deduced Eco57I endonuclease and methylase amino acid sequences revealed three regions of significant similarity. Two of them resemble the conserved sequence motifs characteristic of the DNA[adenine-N6] methylases. The third one shares similarity with corresponding regions of the PaeR7I, TaqI, CviBIII, PstI, BamHI and HincII methylases. Homologs of this sequence are also found within the sequences of the PaeR7I, PstI and BamHI restriction endonucleases. This is the first example of a family of cognate restriction endonucleases and methylases sharing homologous regions. Analysis of the structural relationship suggests that the type IV enzymes represent an intermediate in the evolutionary pathway between the type III and type II enzymes.