Functional screening of a soil metagenome for DNA endonucleases by acquired resistance to bacteriophage infection
Authors: Konanani Rashamuse
Sibongile Mtimka
Samantha Gildenhuys
Tsepo L Tsekoa
Priyen Pillay
Journal: Molecular Biology Reports (2020)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: Bacteriophage; Fosmid library; Functional screening; Kogelberg nature reserve restriction endonuclease; Restriction enzymes
Affiliations: Biomanufacturing Technologies, CSIR Future Production: Chemicals, P O Box 395, Pretoria, 0001, South Africa., Department of Life & Consumer Sciences, College of Agriculture & Environmental Sciences, University of South Africa, Florida Campus (The Science Hub), Florida, P O Box 392, Johannesburg, South Africa., Biomanufacturing Technologies, CSIR Future Production: Chemicals, P O Box 395, Pretoria, 0001, South Africa. .

Abstract:  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. 

Retracted: No

PubPeer comments:
 Authors approached a very important and quickly developing field of phage defence enzymes systems with a novel functional metagenomics approach, which potentially can be very perspective in detecting gene clusters not easily discoverable with current bioinformatics methods. However, there are significant drawbacks in this work, in particular the lack of demonstrated controls, that does not allow to fully believe in reported results. Please consider at least these 2 points. 1. Selection of inserts nuclease genes that grant phage resistance. The easiest way in which bacteria can acquire phage resistance is through mutations in the receptor or cell surface modulation, such mutants of *Escherichia coli* can be easily selected in an experiment described by authors with phage u03bb. To demonstrate that phage resistance was acquired through the expression of defence genes from the received fosmid with metagenomic DNA insert, and not through the mutation in the host chromosome, the fosmid insert from individually selected clones should be transmitted to the wild-type *E. coli* background. The same argument is applicable to the selection of Endo52 nuclease for further work. The authors did not demonstrate that it was Endo52 gene cluster that governed *E. coli* protection against u03bb phage infection, otherwise, it might be just a random nuclease found in metagenomic data. Did the expression of Endo52 full gene cluster from the vector used for protein purification also provide the defence against phage u03bb infection? 2. Functionality of Endo52 nuclease. Authors propose that Endo52 is a functional Type I Restriction-Modification enzyme. The enzymes of this class usually are active as heteropentamers comprised of HsdR, HsdS and HsdM subunits. It is not particularly clear if the expression vector with his-tagged HsdR subunit also contained *hsdS* and *hsdM* genes and if the authors were able to purify the whole complex to obtain a potentially active nuclease enzyme. In figure 3 we observe only the major band suggestively corresponding to the HsdR subunit, which should not be active on its own. There is a probability that minor bands also seen on gel could correspond to the co-purified HsdS and HsdM subunits, but this needs to be proved. ![file] Next, the nuclease activity assay leaves a lot of questions. The time-dependent DNA shmearing pattern can be observed in the case of DNA incubation with any *E. coli* lysate and the degradation of DNA can be associated with a plethora of non-specific host nucleases, having nothing in common with R-M defence systems. Although this could not serve as a final proof for activity, authors could at least demonstrate controls without the addition of SAM and ATP, which would make suggestive Endo52 activity impossible (if this enzyme functions as a classical Type I R-M system). The better proof would be to obtain a nuclease deficient mutant of HsdR subunit and demonstrate a decrease in nuclease activity. At this stage, the presented result suggests that we observe only non-specific activity associated with host nucleases and not an Endo52 activity. ![file] ,