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Restriction–modification (R-M) systems form a large superfamily constituting bacterial innate immunity mechanism. The restriction endonucleases (REases) are very diverse in subunit structure, DNA recognition, co-factor requirement, and mechanism of action. Among the different catalytic motifs, HNH active sites containing REases are the second largest group distinguished by the presence of the ββα-metal finger fold. KpnI is the first member of the HNH-family REases whose homologs are present in many bacteria of Enterobacteriaceae having varied degrees of sequence similarity between them. Considering that the homologs with a high similarity may have retained KpnI-like properties, while those with a low similarity could be different, we have characterized a distant KpnI homolog present in a pathogenic Klebsiella pneumoniae NTUH K2044. A comparison of the properties of KpnI and KpnK revealed that despite their similarity and the HNH motif, these two enzymes have different properties viz oligomerization, cleavage pattern, metal ion requirement, recognition sequence, and sequence specificity. Unlike KpnI, KpnK is a monomer in solution, nicks double-stranded DNA, recognizes degenerate sequence, and catalyses the degradation of DNA into smaller products after the initial cleavage at preferred sites. Due to several distinctive properties, it can be classified as a variant of the Type IIS enzyme having nicking endonuclease activity.Key points• KpnK is a distant homolog of KpnI and belongs to the ββα-metal finger superfamily.• Both KpnI and KpnK have widespread occurrence in K. pneumoniae strains.• KpnK is a Type IIS restriction endonuclease with a single-strand nicking property.

DNA methylation is a key epigenetic alteration that regulates gene expression and plays a vital role in numerous biological processes. Abnormal DNA methylation can lead to various diseases, including cancer. Thus, accurate detection of DNA methylation is crucial for biomedical research. However, conventional methods for methylation detection in clinical settings are slow and tedious. Hence, there is a need for the development of new detection techniques. Fluorescence-based and colorimetric methods have become popular due to their simplicity and rapid response. Furthermore, restriction endonucleases recognise specific DNA sequences and cut the DNA, and are known as the “molecular scissors” of DNA. Methylation-associated restriction endonucleases can selectively cleave DNA based on its methylation status, thereby determining if the target gene is methylated or not. Integration of methylation-associated restriction endonucleases with fluorescence or colorimetric assays offers a fresh approach to DNA methylation detection. This paper reviews traditional detection methods and their drawbacks, emphasizes the use of restriction endonuclease-based fluorescence and colorimetric assays in DNA methylation detection, and discusses existing limitations of these methods along with potential enhancement strategies.

BACKGROUND: Members of the genus Klebsiella are among the leading microbial pathogens associated with nosocomial infection. The increased incidence of antimicrobial resistance in these species has propelled the need for alternate/combination therapeutic regimens to aid clinical treatment. Bacteriophage therapy forms one of these alternate strategies. METHODS: Electron microscopy, burst size, host range, sensitivity of phage particles to temperature, chloroform, pH, and restriction digestion of phage DNA were used to characterize Klebsiella phages. RESULTS AND CONCLUSIONS: Of the 32 isolated phages eight belonged to the family Myoviridae, eight to the Siphoviridae whilst the remaining 16 belonged to the Podoviridae. The host range of these phages was characterised against 254 clinical Enterobacteriaceae strains including multidrug resistant Klebsiella isolates producing extended-spectrum beta-lactamases (ESBLs). Based on their lytic potential, six of the phages were further characterised for burst size, physicochemical properties and sensitivity to restriction endonuclease digestion. In addition, five were fully sequenced. Multiple phage-encoded host resistance mechanisms were identified. The Siphoviridae phage genomes (KP16 and KP36) contained low numbers of host restriction sites similar to the strategy found in T7-like phages (KP32). In addition, phage KP36 encoded its own DNA adenine methyltransferase. The φKMV-like KP34 phage was sensitive to all endonucleases used in this study. Dam methylation of KP34 DNA was detected although this was in the absence of an identifiable phage encoded methyltransferase. The Myoviridae phages KP15 and KP27 both carried Dam and Dcm methyltransferase genes and other anti-restriction mechanisms elucidated in previous studies. No other anti-restriction mechanisms were found, e.g. atypical nucleotides (hmC or glucosyl hmC), although Myoviridae phage KP27 encodes an unknown anti-restriction mechanism that needs further investigation.

Mycobacterium tuberculosis (Mtb), as a typical intracellular pathogen, possesses several putative restriction–modification (R-M) systems, which restrict exogenous DNA’s entry, such as bacterial phage infection. Here, we investigate Rv2528c, a putative Mrr-like type IV restriction endonuclease (REase) from Mtb H37Rv, which is predicted to degrade methylated DNA that contains m6A, m5C, etc. Rv2528c shows significant cytotoxicity after being expressed in Escherichia coli BL21(DE3)pLysS strain. The Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) assay indicates that Rv2528c cleaves genomic DNA in vivo. The plasmid transformation efficiency of BL21(DE3)pLysS strain harboring Rv2528c gene was obviously decreased after plasmids were in vitro methylated by commercial DNA methyltransferases such as M.EcoGII, M.HhaI, etc. These results are consistent with the characteristics of type IV REases. The in vitro DNA cleavage condition and the consensus cleavage/recognition site of Rv2528c still remain unclear, similar to that of most Mrr-family proteins. The possible reasons mentioned above and the potential role of Rv2528c for Mtb were discussed.

Restriction endonucleases are a component of restriction–modification systems, where the main biological function is to protect bacterial cells from incoming foreign DNA molecules. There are four main types of restriction enzymes (types I, II, III, and IV), which differ in protein composition, cofactor requirements, and mode of action. The most studied are representatives of type II, which specifically recognize DNA sequences of 4–8 bp and catalyze DNA cleavage within these sequences or not far from them. The exceptional precision of type II enzymes has made them indispensable for DNA manipulations. Although hundreds of DNA restriction enzymes are currently known, there is still a need for enzymes that recognize new DNA targets. For this reason, the discovery of new natural restriction endonucleases and rational design of their properties (to obtain enzymes with high specificity for a unique nucleotide sequence at a restriction site and without nonspecific activity) will expand the list of enzymes for use in biotechnology and genetic engineering. This review briefly touches upon the main types of restriction endonucleases, their classification, nomenclature, and typical properties, and it concisely describes approaches to the construction of enzymes with altered properties.

The use of rrs (16S rRNA) gene is widely regarded as the “gold standard” for identifying bacteria and determining their phylogenetic relationships. Nevertheless, multiple copies of this gene in a genome is likely to give an overestimation of the bacterial diversity. In each of the 50 Streptococcus genomes (16 species, 50 strains), 4–7 copies of rrs are present. The nucleotide sequences of these rrs genes show high similarity within and among genomes, which did not allow unambiguous identification. A genome-wide search revealed the presence of 27 gene sequences common to all the Streptococcus species. Digestion of these 27 gene sequences with 10 type II restriction endonucleases (REs) showed that unique RE digestion in purH gene is sufficient for clear cut identification of 30 genomes belonging to 16 species. Additional gene-RE combinations allowed identification of another 15 strains belonging to S. pneumoniae, S. pyogenes, and S. suis. For the rest 5 strains, a combination of 2 genes was required for identifying them. The proposed strategy is likely to prove helpful in proper detection of pathogens like Streptococcus.

Endonucleases play a crucial role as reagents in laboratory research and diagnostics. Here, metagenomics was used to functionally screen a fosmid library for endonucleases. A fosmid library was constructed using metagenomic DNA isolated from soil sampled from the unique environment of the Kogelberg Nature Reserve in the Western Cape of South Africa. The principle of acquired immunity against phage infection was used to develop a plate-based screening technique for the isolation of restriction endonucleases from the library. Using next-generation sequencing and bioinformatics tools, sequence data were generated and analysed, revealing 113 novel open reading frames (ORFs) encoding putative endonuclease genes and ORFs of unknown identity and function. One endonuclease designated Endo52 was selected from the putative endonuclease ORFs and was recombinantly produced in Escherichia coli Rosetta™ (DE3) pLysS. Endo52 was purified by immobilised metal affinity chromatography and yielded 0.437 g per litre of cultivation volume. Its enzyme activity was monitored by cleaving lambda DNA and pUC19 plasmid as substrates, and it demonstrated non-specific endonuclease activity. In addition to endonuclease-like genes, the screen identified several unknown genes. These could present new phage resistance mechanisms and are an opportunity for future investigations.

Bacterial identification on the basis of the highly conserved 16S rRNA ( rrs ) gene is limited by its presence in multiple copies and a very high level of similarity among them. The need is to look for other genes with unique characteristics to be used as biomarkers. Fifty-one sequenced genomes belonging to 10 different Yersinia species were used for searching genes common to all the genomes. Out of 304 common genes, 34 genes of sizes varying from 0.11 to 4.42 kb, were selected and subjected to in silico digestion with 10 different Restriction endonucleases (RE) (4–6 base cutters). Yersinia species have 6–7 copies of rrs per genome, which are difficult to distinguish by multiple sequence alignments or their RE digestion patterns. However, certain unique combinations of other common gene sequences— carB , fadJ , gluM , gltX , ileS , malE , nusA , ribD , and rlmL and their RE digestion patterns can be used as markers for identifying 21 strains belonging to 10 Yersinia species: Y. aldovae , Y. enterocolitica , Y. frederiksenii , Y. intermedia , Y. kristensenii , Y. pestis , Y. pseudotuberculosis , Y. rohdei , Y. ruckeri , and Y. similis. This approach can be applied for rapid diagnostic applications.

Diversity analysis of Clostridium botulinum strains is complicated by high microheterogeneity caused by the presence of 9–22 copies of rrs (16S rRNA gene). The need is to mine genetic markers to identify very closely related strains. Multiple alignments of the nucleotide sequences of the 212 rrs of 13 C. botulinum strains revealed intra- and inter-genomic heterogeneity. Low intragenomic heterogeneity in rrs was evident in strains 230613, Alaska E43, Okra, Eklund 17B, Langeland, 657, Kyoto, BKT015925, and Loch Maree. The most heterogenous rrs sequences were those of C. botulinum strains ATCC 19397, Hall, H04402065, and ATCC 3502. In silico restriction mapping of these rrs sequences was observable with 137 type II Restriction endonucleases (REs). Nucleotide changes (NC) at these RE sites resulted in appearance of distinct and additional sites, and loss in certain others. De novo appearances of RE sites due to NC were recorded at different positions in rrs gene. A nucleotide transition A>G in rrs of C. botulinum Loch Maree and 657 resulted in the generation of 4 and 10 distinct RE sites, respectively. Transitions A>G, G>A, and T>C led to the loss of RE sites. A perusal of the entire NC and in silico RE mapping of rrs of C. botulinum strains provided insights into their evolution. Segregation of strains on the basis of RE digestion patterns of rrs was validated by the cladistic analysis involving six house keeping genes: dnaN, gyrB, metG, prfA, pyrG, and Rho.

Bacterial identification using rrs (16S rRNA) gene is widely reported. Bacteria possessing multiple copies of rrs lead to overestimation of its diversity. Staphylococcus genomes carries 5–6 copies of rrs showing high similarity in their nucleotide sequences, which lead to ambiguous results. The genomes of 31 strains of Staphylococcus representing 7 species were searched for the presence of common genes. In silico digestion of 34 common genes using 10 restriction endonucleases (REs) lead to select gene-RE combinations, which could be used as biomarkers. RE digestion of recA allowed unambiguous identification of 13 genomes representing all the 7 species. In addition, a few more genes (argH, argR, cysS, gyrB, purH, and pyrE) and RE combinations permitted further identification of 12 strains. By employing additional RE and genes unique to a particular strain, it was possible to identify the rest 6 Staphylococcus aureus strains. This approach has the potential to be utilized for rapid detection of Staphylococcus strains.