Promiscuous restriction is a cellular defense strategy PNAS PLUS that confers fitness advantage to bacteria Kommireddy Vasua, Easa Nagamalleswaria, and Valakunja Nagarajaa,b,1 aDepartment of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India; and bJawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560064, India Edited by Werner Arber, der Universitat Basel, Basel, Switzerland, and approved March 20, 2012 (received for review November 22, 2011) Most bacterial genomes harbor restriction–modification systems, ognition and cofactor utilization, whereas the cognate MTase is encoding a REase and its cognate MTase. On attack by a foreign very site-specific (14). In addition to its recognition sequence, DNA, the REase recognizes it as nonself and subjects it to restric- GGTACC, the enzyme binds and cleaves a number of noncanoni- tion. Should REases be highly specific for targeting the invading cal sequences (i.e., TGTACC, GTTACC, GATACC, GGAACC, foreign DNA? It is often considered to be the case. However, when GGTCCC, GGTATC, GGTACG, GGTACT), with the binding bacteria harboring a promiscuous or high-fidelity variant of the affinity ranging from 8 to 35 nM (14, 15). Accordingly, many of fi REase were challenged with bacteriophages, tness was maximal these sites are cleaved efficiently with kcat values varying from 0.06 − − under conditions of catalytic promiscuity. We also delineate pos- to 0.15 min 1 vs. 4.3 min 1 for canonical sites (15). The promis- sible mechanisms by which the REase recognizes the chromosome cuous activity of the enzyme is directed by a number of cofactors. as self at the noncanonical sites, thereby preventing lethal dsDNA In the presence of the physiological levels of Mg2+, the most breaks. This study provides a fundamental understanding of how abundant cofactor present in the cell (16) and typically required bacteria exploit an existing defense system to gain fitness advan- for the activity of REases, R.KpnI discriminates poorly between tage during a host–parasite coevolutionary “arms race.” the canonical and noncanonical sequences, indicating that the promiscuity is an intrinsic property of the enzyme (15). It is in- evolution | KpnI | promiscuous activity | DNA cleavage | triguing why promiscuity is manifested and what would be its bi- genome protection ological significance for the organism. Is it an evolutionary event that remained vestigial, or was it retained with a purpose? The MICROBIOLOGY olecular recognition between enzymes and their substrates present study examines the in vivo manifestation of promiscuity Mand/or the cofactors govern physiological functions. Spec- and the conditions that drive it. We provide evidence for the se- ificity in substrate recognition is considered crucial, whereas lective advantage for bacteria in retention of the promiscuous promiscuity is often associated with suboptimal catalytic prop- activity because it confers additional protection against the in- erties. Typically, active site residues are involved during pro- vading genomes. Targeting the incoming genetic elements at the miscuous catalytic activity, and the mechanism of catalysis used is noncanonical sites counters the two common antirestriction similar, despite flexibility in substrate occupancy (1). It has long strategies, namely, modification of phage genome and decrease in been known that many enzymes exhibit flexibility in substrate R-M recognition sites, and highlights a winning situation for recognition (2, 3). The high specificity, however, is considered bacteria in the evolutionary “arms race” between them and a cornerstone of enzyme catalysis, and attempts have often been their parasites. made to increase the fidelity in vitro by either directed evolution or site-directed mutagenesis (1, 4–6). Although promiscuity is Results thought to play a role in divergence of enzyme function (7), re- In Vivo Promiscuous Activity Confers Better Protection. Given the tention of broad specificity in nature, as opposed to the high robust in vitro promiscuous activity of R.KpnI beyond a con- 2+ specificity for many enzymes, continues to be a paradox. centration of 1 mM Mg (17), we considered its potential to Restriction–modification (R-M) systems are one of the largest cleave noncanonical DNA sequences in vivo. The total in- 2+ groups of enzymes that exhibit a high degree of sequence spec- tracellular Mg concentration could vary from 5–10 mM, with ificity for their target sequences. The components of the R-M the free intracellular concentration reaching up to 2 mM (16, systems, namely, REase and MTase, show wide diversity vis-à-vis 18), which is sufficient to elicit promiscuous cleavage. The REase their recognition patterns, active site architecture, and reaction isolated from Klebsiella pneumoniae, which harbors the KpnI R- mechanisms (8). The REases recognize and cleave specific M system, was tested for its activity on noncanonical DNA dsDNA sequences that are extraneous, whereas the MTase ac- substrates. R.KpnI from the native source exhibited biochemical tivity transfers a methyl group to the same DNA sequence within properties similar to those of the enzyme overexpressed in the host’s genome to allow discrimination between self and Escherichia coli. Notably, promiscuous DNA cleavage was ob- nonself DNA. R-M systems thus serve as primitive “innate” served (Fig. 1A), indicating the inherent promiscuous nature of immune systems that provide 102-to108-fold protection for the the REase. host cell against invading bacteriophages and other genetic ele- To investigate a potential role for in vivo promiscuous activity, ments (9, 10). The innateness is attributed to the high specificity we took advantage of a point mutant of R.KpnI, D163I, which of the REases, which cleave the foreign DNA at the canonical exhibits high-fidelity (HF) DNA cleavage (17). The HF variant did sites several orders of magnitude more readily than at the non- canonical sequences. The immaculate specificity achieved by REases has been a subject of extensive study using biochemical, Author contributions: K.V. and V.N. designed research; K.V. and E.N. performed research; thermodynamic, and structural approaches (8, 11–13). Physio- K.V. and V.N. analyzed data; and V.N. wrote the paper. logically, the exquisite specificity is considered an important The authors declare no conflict of interest. virtue of these enzymes to target the invading genomes better. This article is a PNAS Direct Submission. Studies carried out with R.KpnI, a type II REase, opened up 1To whom correspondence should be addressed. E-mail: [email protected]. a different perspective on this prevailing theme, because the en- See Author Summary on page 7608 (volume 109, number 20). zyme exhibits highly promiscuous cleavage under certain conditions This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (14). The REase exhibits remarkable versatility in substrate rec- 1073/pnas.1119226109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1119226109 PNAS | Published online April 16, 2012 | E1287–E1293 Downloaded by guest on October 2, 2021 -GGTACC- -GaTACC- 15 A B GGTACC GaTACC WT WT C 10 HF HF 20 mer 12 mer 5 Product (nM) Product 0 50 100 150 200 Time (sec) C PFU (WT) =1 Comparable in vivo Similar PFU (HF) activity protection Better PFU (WT) <1 WT exhibits in vivo protection PFU (HF) promiscuous activity by WT D 101 100 Strain PFU/ml 10-1 Vector 5.2 x109 10-2 WT 4.2 x105 -3 10 HF 6.0 x107 10-4 H149A 5.4 x109 10-5 Relative plaque forming units T F A H ctor W Ve H149 Fig. 1. Promiscuous activity of KpnI REase confers better protection. (A) Oligonucleotides (10 nM, 5′ end-labeled) containing a canonical sequence (-GGTACC-) or one of the most preferred noncanonical sequences (-GaTACC-) were incubated with Mg2+ and different dilutions of cell-free extracts of K. pneumoniae strain OK8 for 30 min at 37 °C and analyzed on an 8 M urea – 12% (wt/vol) polyacrylamide gel. The enzyme-mediated DNA cleavage of the 20-mer substrate generates a 12-mer end-labeled product. Lane C is substrate DNA with no enzyme. (B) Rates of DNA cleavage by WT and HF enzymes were assayed in the presence of canonical and noncanonical substrates. Reactions contained 1 nM (for canonical DNA cleavage) or 15 nM (for noncanonical DNA cleavage) of the enzyme and 100 nM of substrate in 10 mM Tris·HCl (pH 7.4). The reactions were initiated by the addition of 2 mM Mg2+ and incubation at 37 °C for different time intervals. The plot depicts the product formed vs. time. Data are presented as mean ± SEM. (C) Experimental design of the phage titration assay. Plaque-forming units (PFU) with cells harboring WT and HF were compared. A similar PFU count on both of the strains would indicate a comparable restriction by the WT and HF variants. Alternatively, a lower PFU count on WT-harboring cells compared with the HF variant would indicate greater protection against phages. (D)P1vir phage restriction by R.KpnI and its variants. The plaque-forming units of P1vir phage on cells harboring the WT, the HF variant, or the catalysis-deficient mutant (H149A) relative to cells containing empty vector are shown. (Left) Values from two independent experiments conducted in quadruplicate are plotted. (Right) Representative titer values are shown. not show any promiscuous behavior even at high enzyme or Mg2+ strategies to evade restriction by host REases. One of these strat- concentrations but exhibited a cleavage rate comparable to the egies is to decrease effective REase recognition sites in the genome WT enzyme at the canonical sequences (Fig. 1B and Fig. S1). either by accumulation of point mutations or by acquisition of DNA Because the primary function of R-M systems is to restrict the modification genes (10, 20, 21). Because phage restriction efficiency xenogeneic DNA and protect the host from potent invading life of a REase is directly proportional to the number of its recognition forms, such as bacteriophages (19, 20), phage titer analysis gives an sites in the genome, these antirestriction strategies would allow in vivo measurement of the REase activity (Fig.