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 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 , 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- | KpnI | promiscuous activity | DNA cleavage | triguing why promiscuity is manifested and what would be its bi- 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 . 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 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. 1C). To investigate perpetuation of the foreign DNA in bacteria and increase the fit- whether the recognition of noncanonical sequences confers better ness of the phage (22). If that is the case, cells harboring an HF vir protection against phages, P1 phage was used as an infectious variant would no longer be effective in restricting the modified E. coli + genome element and BL26 (DE3) MK strain harboring phage, whereas the promiscuous WT REase would still be effective either R.KpnI WT (pETRK-WT) or HF (pETRK-HF) was used as fi and decrease the EOP. a host (Table 1). Phage titer assays revealed that the ef ciency of To test the prediction, E. coli BL26 (DE3) MK+ cells harboring plating (EOP) on strains harboring WT or HF enzymes was two to WT (pETRK-WT) or HF (pETRK-HF) variant REase were four orders of magnitude lower than on those lacking the REase or vir fi fi challenged with phage P1 modi ed at canonical sequences harboring a catalytically de cient mutant of REase, suggesting ef- (Materials and Methods). The modified phage (P1*) is poorly re- fective restriction of the invading phage by R.KpnI (Fig. 1D). stricted by the cells harboring the HF REase (Fig. 2). In contrast, However, a two orders of magnitude better restriction of the the P1* population was reduced to <1% by the strains harboring infecting phage DNA is observed with cells containing WT REase fi compared with the HF enzyme. A more effective restriction of the WT enzyme, indicating that the relaxed cleavage speci city the phage by WT could be attributed to either of two reasons: The of R.KpnI confers additional protection against the invading enzyme exhibits cleavage at noncanonical sites in addition to the genomes. To determine whether the observed features are in- canonical sequences, or it possesses higher activity at canonical dependent of the number of KpnI sites in the foreign genome(s), λvir sequences compared with the HF variant. Kinetic analysis with titrations were carried out with the ,whichcon- the WT and HF variants of R.KpnI revealed that the enzymes tains only two GGTACC sites in the genome. The analysis with the possess a comparable turnover value for the canonical recognition λvir (unmodified) and λ*(modified) substantiated the role of sites (Table 2). This suggested that the WT REase recognizes promiscuous activity in providing a fitness advantage to bacteria in additional sequences on the phage genome. a phage-enriched environment (Table S1). To provide further support for our hypothesis that catalytic Promiscuous Activity Counters the Antirestriction Strategies. Bac- promiscuity induced by physiological levels of the REase results teriophages and other invasive genome elements use several in more proficient restriction of phages, titrations were carried

E1288 | www.pnas.org/cgi/doi/10.1073/pnas.1119226109 Vasu et al. Downloaded by guest on October 2, 2021 Table 1. Plasmids and strains used in this study PNAS PLUS Plasmids/strains Characteristics

pETRK-WT R.KpnI cloned into pET11d between NcoI and BamHI sites pETRK-HF R.KpnI D163I cloned into pET11d between NcoI and BamHI sites pACMK M.KpnI cloned into pACYC between EcoRV and HindIII pUCMK M.KpnI cloned into pUC18 between HincII and HindIII pACR-M KpnI R-M system with its own promoter elements cloned into pACYC between HindIII and BamHI pEcHNS E. coli hns cloned into pET16b pEcHU E. coli hupA and hupB cloned into pRLM118 pEcFIS E. coli fis cloned into pUHE pUC18 Plasmid containing a single KpnI site (GGTACC) pUCΔK pUC18 plasmid lacking the KpnI site (generated by deleting the KpnI site from pUC18 by digesting with EcoRI and HindIII) pBR322 Plasmid devoid of KpnI sites K. pneumoniae strain OK8 American Type Culture Collection 4970 strain containing KpnI R-M system E. coli DH10B Δ(mrr-hsd rms-mcrBC) mcrA recA1 E. coli BL26(DE3) F-ompT gal [dcm] [lon] hsdSB (rB-, mB-) Δlac (DE3) nin5 lacUV5-T7 gene 1 E. coli DH10B MK(+) E. coli DH10B harboring pACMK plasmid E. coli BL26(DE3) MK(+) E. coli BL26(DE3) harboring pACMK plasmid Bacteriophage P1vir E. coli phage containing 25 sites for KpnI on the genome Bacteriophage λvir E. coli phage containing 2 sites for KpnI on the genome

out in an E. coli DH10B strain containing a low-copy-number undergo phage-induced cell lysis for up to 5 h, irrespective of plasmid that harbors a kpnI R-M system (Table 1) with its own methylation status of the phage. These results correlated with promoter and regulatory elements. The genes for KpnI REase the EOP observed for P1vir and P1* phage on cells harboring and MTase are arranged divergently and separated by a 167-bp vector, WT enzyme, or HF enzyme, substantiating that pro- MICROBIOLOGY intergenic region that contains all the regulatory elements re- miscuity increases fitness advantage during a phage encounter. quired for the expression of both the genes (23) (Fig. 3A). The promoters are separated by 57 bp and are present in opposite Self vs. Nonself Recognition. The dsDNA breaks catalyzed by strands having typical characteristic features of σ 70 promoters REases could be lethal to the host genome if they are not con- of E. coli (24, 25). The REase expression with its native promoter trolled (26). The host cell harboring promiscuous R.KpnI and in the low-copy plasmid was comparable to that of its level in site-specific M.KpnI (14) needs to protect its genome not only at K. pneumoniae strain OK8 (Fig. 3B), and the strain could restrict the canonical sites but at the noncanonical sites. In case of type I P1vir phage methylated at GGTACC sites (Fig. 3C). The low R-M systems, it is established that the resident chromosome is transformation efficiency of the strain with pBR322, which lacks recognized as self rather than foreign even in the absence of the R.KpnI recognition site (Fig. S2), confirms that the endog- modification, a phenomenon known as “restriction alleviation” enous levels of the REase are sufficient for promiscuous activity (27, 28). However, similar mechanisms are not known for the against foreign genomes. type II R-M systems. Genomes of γ-proteobacteria are com-

Catalytic Versatility Confers Fitness Advantage. The ability of the enzyme to recognize and cleave noncanonical sequences in vivo confers additional protection to the host against the modified A Methylation of GGTACC infectious genome elements. To investigate whether this en- sites in the genome hanced resistance conferred by the promiscuity increases bacte- P1 -phage P1* phage rial survival, growth studies were carried out in the presence of phages (Fig. 4). When E. coli BL26 (DE3) MK+ strains har- B boring vector (pET11d) or constructs encoding WT (pETRK- 10 1 WT) or HF (pETRK-HF) enzyme were challenged with unme- 10 0 thylated (P1vir) and methylated (P1*) phages, cells containing 10 -1 EOP=1 only the vector lysed readily (Fig. 4 A and B). Cells expressing 10 -2

the HF variant undergo phage-induced cell lysis with different 10 -3 EOP=1 EOP<1 degrees of susceptibility. With phage P1vir (unmodified) in- 10 -4 fection, delayed lysis is observed compared with phage P1* -5 (modified). In contrast, WT enzyme-harboring cells did not Relative plaque forming units 10 No Promiscuous r Promiscuous activity as WT HF ecto V H149A activity defense strategy

Table 2. Kinetic parameters of WT and HF variants Fig. 2. Promiscuous activity counters the antirestriction strategies. (A)Ex- − − − perimental design of phage titration assay with modified phage. Phage P1* K ,nM k ,min 1 k /K ,M 1·s 1* M cat cat M (methylated at GGTACC sites in the genome) was prepared with the KpnI Substrates WT HF WT HF WT HF MTase overexpression strain as described in Materials and Methods. The HF variant should not restrict the modified phage, whereas the cells harboring (-GGTACC-) 18 22 4.56 3.38 4.2 (±0.3) × 106 2.5 (±0.5) × 106 a promiscuous WT REase would still decrease the EOP. (B) Plaque-forming fi (-Ga/tTACC-) 53 nd 0.16 nd 5.0 (±0.8) × 104 nd units of phage P1* on cells harboring the WT, HF, or catalysis-de cient mutant (H149A) relative to cells harboring empty vector are shown. The nd, no detectable cleavage. measurements from two separate experiments conducted in quadruplicate *Values are determined from three measurements and are shown as mean ± SE. are plotted.

Vasu et al. PNAS | Published online April 16, 2012 | E1289 Downloaded by guest on October 2, 2021 A sites in it in a processive manner (33, 34). In the case of R.KpnI, a plasmid containing a single recognition site was restricted effi- M.KpnI 167bp R.KpnI ciently at both the canonical and noncanonical sites compared with the plasmid devoid of the canonical site (Fig. 6 A and B). The en- hancement of cleavage at noncanonical sites by the presence of -35 -10 SD a canonical site could occur for two reasons. The enzyme may use M.KpnI -TTTTCA- 17bp -TATAAT- 11bp - AGGAGGGTACATG a canonical site for initial recognition of foreign genome, which is then followed by cleavage at promiscuous sites. Alternatively, be- -TTAAAA- 17bp -AATGTA- 18bp -AACGAGGT ATG R.KpnI ATCAA cause of the presence of a canonical site, the enzyme could rapidly convert the supercoiled substrate into a linear form that can now be B KpOK8 KpnI R-M acted on even at noncanonical sites. In other words, the enzyme M C1 C2 C3 may discriminate sequences through their topological status. To fi R.KpnI verify these possibilities, the ef ciency of promiscuous DNA cleavage was compared with supercoiled and linearized plasmid substrates devoid of canonical sites. The linear DNA served as an 1 2 3 4 5 6 7 efficient substrate compared with the supercoiled DNA (Fig. 6 B– E), indicating that the R.KpnI could discriminate between the self C 10 1 and nonself at noncanonical sites through their topological status. Discussion

10 0 REases are the genomes’ sentinels in bacteria to counter the threat of foreign DNA. Invading genomes evade restriction by using a multitude of antirestriction strategies to increase their 10-1 fitness (10, 20, 35). Bacteria counteract these antirestriction strategies by acquiring additional R-M systems with distinct recognition specificities (36). The continuous selection for the

Relative plaque forming units forming plaque Relative 10-2 fitness advantage leads to a coevolutionary arms race between

-M the phages and their hosts (10, 20). The restriction and anti- I R n restriction genes are thus under a constant evolutionary pressure KpnI M p K to tinker with their functionality for providing a better fitness advantage to respective genomes where they reside. Studies Fig. 3. In vivo analysis of KpnI R-M system-containing cells. (A) Organization of KpnI R-M system. (B) Western blot analysis with R.KpnI polyclonal anti- described here reveal a hitherto undescribed paradigm to bodies. In lane 1, 0.2 μg of purified R.KpnI was used as a marker (M). In lanes counter the antirestriction strategies of the invading genomes. 2–4, increasing amounts (250, 500, and 750 μg) of total from K. Although the specific cleavage by a REase allows bacteria to pneumoniae strain OK8 cell lysate (KpOK8) were used. In lanes 5–7, cell adapt to new environments, its maintenance appears to be se- lysates (250 μg) prepared from three individual clones of E. coli cells lected against in a constantly evolving environment enriched with expressing both MTase and REase from their respective endogenous pro- phages (37). The high specificity has a caveat in inducing adap- moters (KpnI R-M) are shown. (C) Phage P1* titer on E. coli cells harboring tation among phages to evade restriction either by (i) decreasing KpnI MTase alone (KpnI M) or both KpnI MTase and REase (KpnI R-M). The the palindromic sequences (38–40) or (ii) modification (meth- measurements from two separate experiments conducted in quadruplicate ylation or glucosylation) of the phage DNA (10, 20). The two are shown. strategies decrease the effective number of REase recognition sites in the genome of a phage (22, 38–43). The presence of plexed with polyamines and various nucleoid-associated fi multiple R-M systems, adaptive immunity by interference, and (NAPs), all of which bind in a nonspeci c fashion to cause selective silencing of xenogeneic sequences are some of the bending, wrapping, bridging, and/or coating of DNA, which, in – mechanisms that bacteria use to enhance protection against the turn, could affect various DNA transaction processes (29 31). invading foreign DNA (29, 44, 45). Although these bacterio- To determine the mechanisms that operate to discriminate self phage resistance mechanisms offer protection, they operate ei- vs. nonself at the noncanonical sequences, R.KpnI DNA cleav- ther with a low frequency or with lower adaptability (44, 46). The “ ” age activity was compared on naked DNA and DNA coated ability of an enzyme to cleave foreign DNA with broader spec- with polyamines or NAPs. ificity appears to be an improved protection mechanism under When plasmid DNA coated with varying concentrations of these circumstances (Fig. 7). polyamines was used as substrate, promiscuous activity was One of the important attributes of DNA transaction processes suppressed with increasing concentrations of these ligands (Fig. is the ability to distinguish between different DNA substrates, for A 5 ). In the absence of polyamines, the enzyme exhibited robust example, replicated vs. nonreplicated DNA or host vs. foreign promiscuous activity. At 2.5-mM and 0.01-mM concentrations of DNA. For this, the cell has to ensure that the DNA molecules spermidine and spermine, respectively, the promiscuous activity are chemically different as achieved by host DNA methylation. was undetectable. This correlates well with the intracellular When a phage attacks an R-M proficient cell encoding a pro- concentrations of spermidine (6.3 mM) and spermine (<1 mM) miscuous REase and a site-specific MTase, as in the case of the (32). However, at very high concentrations of polyamines, both KpnI R-M system, the host genome and foreign DNA molecules canonical and noncanonical sites were refractory to the enzyme- have to be distinguished by the REase at the noncanonical sites. mediated cleavage (Fig. S3). Similarly, the DNA coated with the The bacterial genome is coated by NAPs and polyamines, and is NAPs (i.e., HU, HNS, Fis) regulated the promiscuous activity to thus recognized by the REase as self (47). Because the foreign varying degrees (Fig. 5B). Suppression of promiscuous activity DNA is likely to be in an uncondensed form (28, 48), it renders was most pronounced in the presence of HNS, followed by that itself the perfect target for the dsDNA cleavage at canonical as of Fis and HU. well as noncanonical sequences. However, the efficiency of re- Next, we investigated additional mechanisms that could operate striction at noncanonical sites would depend on the kinetics of to discriminate self vs. nonself at the noncanonical sequences. the association of invading DNA with polyamines and NAPs. Generally, REases recognize the foreign DNA and cleave target Apart from what is described here for R.KpnI, the ability to dis-

E1290 | www.pnas.org/cgi/doi/10.1073/pnas.1119226109 Vasu et al. Downloaded by guest on October 2, 2021 PNAS PLUS A Presence of phage P1 B Presence of phage P1* 0.60 0.60 Vector Vector WT WT 0.45 0.45 HF HF

0.30 0.30 O.D. O.D.

0.15 0.15

0.00 0.00 0 80 160 240 320 0 80 160 240 320 Time (min) Time (min)

Absence of phage C 2.0 Vector

1.5 WT HF

1.0 O.D.

0.5

0.0 0 100 200 300 400 500 Time (min)

Fig. 4. Catalytic versatility confers fitness advantage. Growth profiles of E. coli BL26 cells harboring vector, the WT, or the HF variant in the presence of P1vir MICROBIOLOGY

(A) or P1* (P1vir phage methylated at KpnI sites) (B). (C) Growth curve analysis of the cells in the absence of phage. Growth was monitored at OD600. Growth curves shown are representative of three independent experiments.

criminate spermine-coated host genome vs. naked foreign DNA antirestriction mechanisms of the phages. The counterstrategies is also reported for the type I enzyme EcoKI (49). Host genome that may potentially be developed by the phages against the protection from R.KpnI seems to be distinct in using both the REase promiscuity, if any, need further investigation. DNA topology and the nucleoid organization to discriminate be- The high degree of promiscuity exhibited by R.KpnI hints at the tween the host vs. xenogeneic genomes at noncanonical sequences. evolutionary link between the nonspecific and highly sequence- Despite all these protection mechanisms, accidental DNA specific . Specific endonucleases have either evolved scissions, if inflicted by the REase, could be rescued by DNA into nonspecific endonucleases or vice versa because of certain ligase or homologous recombination-mediated repair pathways. selection forces. In the case of R.KpnI, although it is possible that These repair mechanisms are known to rescue cells from le- the HF variants of the enzyme could exist in the natural habitat, thality caused by type I and type II REases (50–53). Hence, it the organism appears to prefer promiscuous activity. Moreover, the appears that the cell’s ability to discriminate between its own cofactor Mg2+ that activates promiscuous activity is used by the DNA and foreign DNA on the basis of (i) canonical methylation, enzyme in vivo over other metal ions (Ca2+ or Zn2+) that induce (ii) nucleoid organization, and (iii) differences in DNA topology fidelity in DNA cleavage. We surmise that promiscuous activity of and repair mechanisms protect the cell from detrimental con- R.KpnI has either been retained or has evolved to counteract the sequences of accidental host genome destruction and provide attack by foreign genomes. a fitness advantage. The reason for the occurrence of catalytically proficient, non- Recent studies carried out with >200 REases have revealed canonical DNA cleavage by REases under altered conditions has that a majority of the REases in the group exhibit relaxed sub- been enigmatic. Our study answers an important but unaddressed strate specificity (54, 55). Thus, it appears that the catalytic question as to why a given REase retains catalytic promiscuous promiscuous activity is an intrinsic property of these enzymes. activity. The retention of the promiscuous cleavage characteristics Notably, a single point could convert the promiscuous of a type II REase that is normally expected to possess exquisite R.KpnI to an HF REase (17). Taking a cue from this work, 27 sequence specificity provides a selective advantage for the bacte- promiscuous REases were converted into HF enzymes by point rial genome in the coevolutionary arms race between phages mutations (6). It is likely that many REases retain an inherent and bacteria. flexibility in sequence recognition during the course of evolution. Thus, the current paradigm of high sequence specificity of Materials and Methods REases for their physiological purpose may need to be revisited. Bacterial Strains, Plasmids, Substrates, and Enzymes. The strains and geno- It is unlikely that every type II enzyme would exhibit character- types of bacteria and plasmids used in this study are listed in Table 1. K. istics similar to R.KpnI, given their vast diversity. Nevertheless, pneumoniae strain OK8 (American Type Culture Collection 4970 strain) promiscuity could be a widespread phenomenon, because it may containing a KpnI R-M system was used for assays with cell be a kind of natural law favoring flexibility. extracts. E. coli strains DH10B and BL26 (DE3) were used for cloning and Endonuclease II of T4 coliphage uses relaxed specificity to overexpression, respectively. R.KpnI was expressed in E. coli BL26 cells in the presence of pACMK (plasmid-expressing KpnI MTase from its native pro- initiate host DNA degradation, whereas the phage genome is moter). All inductions were done with 50 μM isopropyl-β-D-thiogalactopyr- protected because of hydroxymethylated cytosines (56). Thus, anoside, and the expression of R.KpnI and its mutants in various in vivo the utilization of promiscuous activity to target the opponent experiments was confirmed by Western blot analysis. The kpnI R-M system genome exists in phages. Now, it is apparent that a similar with its own promoter elements was cloned in pACYC184 at HindIII and strategy could have been developed by bacteria to counteract the BamHI sites (pACR-M) with an additional support plasmid expressing KpnI

Vasu et al. PNAS | Published online April 16, 2012 | E1291 Downloaded by guest on October 2, 2021 Spermidine ABHNS A 0 5 10 15 30 min B 0 5 10 15 30 min C 0 5 10 15 30 min OC OC C 0 0.5 1 2.5 5 10 mM C 0 25 100 250 nM OC OC L L L L Sp L Sp SC SC SC SC P P P P

Spermine Fis pΔ81CU CUp K pUCΔK-Linear C 0 10 25 50 100 500 μM C 0 25 100 250 nM OC OC 0 5 10 15 30 min 0 5 10 15 30 min L Sp L Sp D OC E SC L L SC SC P P

P

Protamine HU C 0 0.5 1 2.5 5 25 μM C 0 25 100 250 nM pBR322 pBR322-Linear OC OC L L Sp Sp Fig. 6. Effect of canonical recognition sequence on the promiscuous activity of SC SC R.KpnI. Plasmids (14 nM) with (pUC18) or without (pUCΔK or pBR322) the KpnI P P recognition sequence were incubated with the enzyme (30 nM) on ice for 10 min. The reactions were then initiated by adding 2 mM Mg2+ to the plasmid enzyme mixture. The reactions were stopped at the indicated time points by adding a stop dye containing 10 mM EDTA. (A) pUC18 DNA containing a single KpnI Fig. 5. Suppression of promiscuous activity of R.KpnI by polyamines and recognition sequence. (B)pUCΔK DNA (pUC18 plasmid lacking the KpnI site). (C) NAPs. Plasmid DNA cleavage activity of R.KpnI was assayed in the presence of Linearized pUCΔK DNA. (D) pBR322 DNA. (E) Linearized pBR322 DNA. P indicates different concentrations of polyamines, namely, spermidine and spermine or promiscuous DNA cleavage products. SC, L, and OC indicate the positions of the NAPs (i.e., HNS, Fis, HU). Protamine, a small arginine-rich protein was used as supercoiled, linear, and open circular forms of the plasmid, respectively. a control. Reactions contained 30 nM R.KpnI, and titration was carried out by preincubation of the supercoiled pUC18 DNA (14 nM) in buffer containing 10 mM Tris·HCl (pH 7.4) or 2 mM Mg2+ on ice with spermidine (0.5, 1, 2.5, 5, and and grown. In the case of growth studies in the presence of phages, ∼106 10 mM), spermine (10, 25, 50, 100, and 500 μM), or protamine (0.5, 1, 2.5, 5, and 25 μM) (A)orNAPs(25,100,and250nM)(B). The reactions were initiated plaque forming units (PFU) were inoculated along with the bacterial cul- by addition of the enzyme and incubation at 37 °C for 1 h. Lane C is substrate tures. Growth was measured by taking absorbance at 600 nm. DNA with no enzyme. Lane 0 shows the DNA cleavage reaction in the absence of polyamines or NAPs. Sp and P indicate specificandpromiscuousDNA cleavage products, respectively. SC, L, and OC indicate the positions of the supercoiled, linear, and open circular forms of the plasmid, respectively. Co-evolutionary ‘arms race’ Winner Phage Bacteria

MTase (pUCMK). The positive clones were cured for the pUCMK plasmid by A No Phage growing the culture in the absence of ampicillin. The cells cured for pUCMK R-M system express both MTase and REase from their respective endogenous promoters fi (KpnI R-M). R.KpnI expression was con rmed by Western blot analysis using Acquisition polyclonal antibodies. The clones with REase expression comparable to its of R-M system level in K. pneumoniae strain OK8 were used for the in vivo assays. Oligonucleotides (Sigma–Aldrich) used for DNA cleavage assays (Table S2) B R-M Bacteria were purified on 8 M urea – 18% (wt/vol) polyacrylamide gel. The purified system oligonucleotides were end-labeled with T4 polynucleotide kinase and [γ-32P] ATP (6,000 Ci/mmol). R.KpnI and its mutants were purified using the method Anti-restriction strategies described previously (25). E. coli HNS was purified with a nickel-nitriloacetic acid-agarose column (Qiagen). E. coli Fisproteinwaspurified as described (57). R-M E. coli HU protein was purified as described (58). The purity of the proteins was C Phage system assessed by SDS/PAGE. Evolution of promiscuous Phage Restriction and Growth Studies. Restriction activity in vivo was estimated cleavage by the ability of cells with an R-M system to restrict plaque formation by P1vir and λvir bacteriophages. P1vir and λvir phage strains possess 25 and 2 sites for D Promiscuous Bacteria KpnI (GGTACC), respectively. The media used for these experiments contained REase selective antibiotics. Modification of the phage genome was carried out by preparing the phage lysates on E. coli BL26 strain overexpressing KpnI MTase Fig. 7. Coevolutionary arms race between the phages and their hosts results in (pUCMK). M.KpnI does not exhibit promiscuous behavior and methylates in a the utilization of promiscuous activity as a defense strategy. Phage as the winner site-specific manner (14). The modification status of the phage genomes indicates successful infection of the host. Bacteria as the winner indicate efficient was confirmed by resistance to site-specific cleavage by KpnI REase both in restriction of phage DNA. (A) In the absence of an R-M system, the phage emerges vitro and in vivo. Phage plaque assays were carried out using the top agar as the winner because of its ability to infect the bacteria. (B) Host adapts by ac- overlay technique. Briefly, an overnight culture of E. coli BL26 MK+ cells har- quiring an R-M system that can now restrict the invading DNA elements. (C)This, boring vector, the WT, or the HF variant was diluted 100-fold and grown to in turn, leads to the development of antirestriction strategies in the phages by (i) midexponential phase at 37 °C with aeration in LB broth. Phages were ap- acquisition of DNA modification systems (e.g., methylation, glucosylation) or (ii) − − propriately diluted (10 3 to 10 7 serial dilutions) and mixed with 100 μLofthe avoiding palindromic DNA sequences. (D) However, a promiscuous REase would fresh culture. After incubation at 37 °C for 30 min, the phage–bacteria com- target even those phages that are equipped with antirestriction mechanisms, plex was mixed with 3 mL of LB top agar and poured on an agar plate. After thus conferring a survival advantage. The promiscuous cleavage characteristics incubation at 37 °C for 8–12 h, plaques were counted. maybeacquiredbythesite-specific REase or retained during the evolution. For growth studies, overnight cultures of E. coli BL26 MK+ cells harboring Irrespective of the directionality, possessing promiscuous activity is advantageous vector, the WT, or the HF variant were diluted (1:500) into fresh LB medium to the bacteria in better restricting the invading genome elements.

E1292 | www.pnas.org/cgi/doi/10.1073/pnas.1119226109 Vasu et al. Downloaded by guest on October 2, 2021 DNA Condensation and in Vitro DNA Cleavage Activity. DNA condensation was harvested by centrifugation, resuspended in 3 mL of extraction buffer [10 PNAS PLUS carried out using different concentrations of polyamines (spermidine and mM Tris·HCl (pH 8.0), 50 mM NaCl, 5 mM 2-mercaptoethanol], and disrupted spermine) and protamine (a small arginine-rich protein was used as a control) by sonication. Cell debris was removed by centrifugation, and dilutions of in 10 mM Tris·HCl (pH 7.4). Stock solutions of spermidine, spermine, and the supernatant (1:1,000 to 1:10) prepared with extraction buffer were used protamine (Sigma–Aldrich) were added to DNA in the reaction buffer and for the DNA cleavage assays as described (14). incubated on ice for 15 min before adding R.KpnI and 2 mM Mg2+. Activity on the modified DNA was analyzed by incubation for 1 h at 37 °C. DNA ACKNOWLEDGMENTS. We thank D. N. Rao and S. Mahadevan for E. coli HU- cleavage assays were carried out using 14 nM pUC18, pUCΔK (pUC18 lacking and HNS-overexpressing clones, P. Uma Maheswari for technical assistance, and fi KpnI site), and pBR322 as described (14). For pUC18, the enzyme/canonical S. Ghosh and S. Karambelkar for puri ed E. coli HU and Fis proteins, respectively. site ratio was 2.1:1, and for all three plasmids used, the enzyme/non- New England Biolabs provided K. pneumoniae strain OK8. We thank T. A. Bickle, K. P. Gopinathan, D. N. Rao, U. Varshney, and members of the V.N. laboratory canonical site ratio was lower than 1. Linearized plasmid substrates of for critical reading of the manuscript. K.V. is the recipient of a Shyama Prasad Δ pUC K and pBR322 were prepared by digesting with R.BamHI. For DNA Mukherjee Fellowship from the Council of Scientific and Industrial Research, cleavage assays with cell extracts, K. pneumoniae strain OK8 containing the Government of India. V.N. is the recipient of a J. C. Bose Fellowship from the KpnI R-M system was grown in 50 mL of LB to an OD600 of 0.6. Cells were Department of Science and Technology, Government of India.

1. Khersonsky O, Roodveldt C, Tawfik DS (2006) Enzyme promiscuity: Evolutionary and 30. Tabor CW, Tabor H (1984) Polyamines. Annu Rev Biochem 53:749–790. mechanistic aspects. Curr Opin Chem Biol 10:498–508. 31. Pingoud A, et al. (1984) Effect of polyamines and basic proteins on cleavage of DNA 2. Jensen RA (1976) Enzyme recruitment in evolution of new function. Annu Rev by restriction endonucleases. Biochemistry 23:5697–5703. Microbiol 30:409–425. 32. Tabor CW, Tabor H (1976) 1,4-Diaminobutane (putrescine), spermidine, and spermine. 3. O’Brien PJ, Herschlag D (1999) Catalytic promiscuity and the evolution of new Annu Rev Biochem 45:285–306. enzymatic activities. Chem Biol 6:R91–R105. 33. Jeltsch A, Urbanke C (2004) Sliding or hopping? How restriction enzymes find their 4. Aharoni A, et al. (2005) The ‘evolvability’ of promiscuous protein functions. Nat Genet way on DNA restriction endonucleases. Nucleic Acids and ,ed 37:73–76. Pingoud AM (Springer, Berlin), Vol 14, pp 95–110. 5. Yoshikuni Y, Ferrin TE, Keasling JD (2006) Designed divergent evolution of enzyme 34. Gowers DM, Halford SE (2003) Protein motion from non-specific to specific DNA by function. Nature 440:1078–1082. three-dimensional routes aided by supercoiling. EMBO J 22:1410–1418. 6. Zhu Z, et al. (2009) Patent US 2009/0029376 A1. 35. Bickle TA (2004) Restricting restriction. Mol Microbiol 51:3–5. 7. Khersonsky O, Tawfik DS (2010) Enzyme promiscuity: A mechanistic and evolutionary 36. Zheng Y, et al. (2010) A unique family of Mrr-like modification-dependent restriction perspective. Annu Rev Biochem 79:471–505. endonucleases. Nucleic Acids Res 38:5527–5534. 8. Pingoud A, Fuxreiter M, Pingoud V, Wende W (2005) Type II restriction endonucleases: 37. Chinen A, Naito Y, Handa N, Kobayashi I (2000) Evolution of sequence recognition by Structure and mechanism. Cell Mol Life Sci 62:685–707. restriction-modification enzymes: Selective pressure for specificity decrease. Mol Biol 9. Arber W, Linn S (1969) DNA modification and restriction. Annu Rev Biochem 38:467–500. Evol 17:1610–1619.

10. Tock MR, Dryden DTF (2005) The biology of restriction and anti-restriction. Curr Opin 38. Karlin S, Mrázek J, Campbell AM (1997) Compositional biases of bacterial genomes MICROBIOLOGY Microbiol 8:466–472. and evolutionary implications. J Bacteriol 179:3899–3913. 11. Cowan JA, ed (2004) Role of Metal Ions in Promoting DNA Binding and Cleavage by 39. Blaisdell BE, Campbell AM, Karlin S (1996) Similarities and dissimilarities of phage restriction Endonucleases (Springer, Berlin), pp 339–360. genomes. Proc Natl Acad Sci USA 93:5854–5859. 12. Thielking V, Alves J, Fliess A, Maass G, Pingoud A (1990) Accuracy of the EcoRI 40. Gelfand MS, Koonin EV (1997) Avoidance of palindromic words in bacterial and restriction endonuclease: Binding and cleavage studies with oligodeoxynucleotide archaeal genomes: A close connection with restriction enzymes. Nucleic Acids Res 25: substrates containing degenerate recognition sequences. Biochemistry 29:4682–4691. 2430–2439. 13. Sapienza PJ, Dela Torre CA, McCoy, WH, 4th, Jana SV, Jen-Jacobson L (2005) 41. Sharp PM (1986) Molecular evolution of bacteriophages: Evidence of selection against Thermodynamic and kinetic basis for the relaxed DNA sequence specificity of the recognition sites of host restriction enzymes. Mol Biol Evol 3:75–83. “promiscuous” mutant EcoRI endonucleases. J Mol Biol 348:307–324. 42. Warren RA (1980) Modified bases in bacteriophage DNAs. Annu Rev Microbiol 34:137–158. 14. Chandrashekaran S, Saravanan M, Radha DR, Nagaraja V (2004) Ca(2+)-mediated site- 43. Swinton D, et al. (1983) Purification and characterization of the unusual deoxynucleoside, specific DNA cleavage and suppression of promiscuous activity of KpnI restriction alpha-N-(9-beta-D-2′-deoxyribofuranosylpurin-6-yl)glycinamide, specified by the phage endonuclease. J Biol Chem 279:49736–49740. Mu modification function. Proc Natl Acad Sci USA 80:7400–7404. 15. Saravanan M, Vasu K, Kanakaraj R, Rao DN, Nagaraja V (2007) R.KpnI, an HNH 44. Brouns SJ, et al. (2008) Small CRISPR RNAs guide antiviral defense in . superfamily REase, exhibits differential discrimination at non-canonical sequences in Science 321:960–964. the presence of Ca2+ and Mg2+. Nucleic Acids Res 35:2777–2786. 45. Cardinale CJ, et al. (2008) Termination factor Rho and its cofactors NusA and NusG 16. Cowan JA (2002) Structural and catalytic chemistry of magnesium-dependent silence foreign DNA in E. coli. Science 320:935–938. enzymes. Biometals 15:225–235. 46. Horvath P, et al. (2008) Diversity, activity, and evolution of CRISPR loci in 17. Saravanan M, Vasu K, Nagaraja V (2008) Evolution of sequence specificity in a restriction Streptococcus thermophilus. J Bacteriol 190:1401–1412. endonuclease by a point mutation. Proc Natl Acad Sci USA 105:10344–10347. 47. Dorman CJ (2009) Nucleoid-associated proteins and bacterial physiology. Adv Appl 18. Alatossava T, Jütte H, Kuhn A, Kellenberger E (1985) Manipulation of intracellular Microbiol 67:47–64. magnesium content in polymyxin B nonapeptide-sensitized Escherichia coli by 48. Luijsterburg MS, White MF, van Driel R, Dame RT (2008) The major architects of ionophore A23187. J Bacteriol 162:413–419. chromatin: Architectural proteins in bacteria, archaea and eukaryotes. Crit Rev 19. Arber W (1965) Host specificity of DNA produced by Escherichia coli V. The role of Biochem Mol Biol 43:393–418. methionine in the production of host specificity. J Mol Biol 11:247–256. 49. Keatch SA, Su TJ, Dryden DTF (2004) Alleviation of restriction by DNA condensation 20. Bickle TA, Krüger DH (1993) Biology of DNA restriction. Microbiol Rev 57:434–450. and non-specific DNA binding ligands. Nucleic Acids Res 32:5841–5850. 21. Krüger DH, Barcak GJ, Smith HO (1988) Abolition of DNA recognition site resistance 50. Cromie GA, Leach DR (2001) Recombinational repair of chromosomal DNA double- to the restriction endonuclease EcoRII. Biomed Biochim Acta 47:K1–K5. strand breaks generated by a restriction endonuclease. Mol Microbiol 41:873–883. 22. Wilson GG, Murray NE (1991) Restriction and modification systems. Annu Rev Genet 51. Heitman J, Zinder ND, Model P (1989) Repair of the Escherichia coli chromosome after 25:585–627. in vivo scission by the EcoRI endonuclease. Proc Natl Acad Sci USA 86:2281–2285. 23. Chatterjee DK, Hammond AW, Blakesley RW, Adams SM, Gerard GF (1991) Genetic 52. Smith MD, Longo M, Gerard GF, Chatterjee DK (1992) Cloning and characterization of organization of the KpnI restriction–modification system. Nucleic Acids Res 19:6505–6509. genes for the PvuI restriction and modification system. Nucleic Acids Res 20:5743–5747. 24. Hawley DK, McClure WR (1983) Compilation and analysis of Escherichia coli promoter 53. Tímár E, Venetianer P, Kiss A (2008) In vivo DNA protection by relaxed-specificity SinI DNA sequences. Nucleic Acids Res 11:2237–2255. DNA methyltransferase variants. J Bacteriol 190:8003–8008. 25. Chandrashekaran S, Babu P, Nagaraja V (1999) Characterization of DNA binding 54. Wei H, Therrien C, Blanchard A, Guan S, Zhu Z (2008) The Fidelity Index provides activities of over-expressed kpnI restriction endonuclease and modification methylase. a systematic quantitation of star activity of DNA restriction endonucleases. Nucleic J Biosci 24:269–277. Acids Res 36:e50. 26. Asakura Y, Kobayashi I (2009) From damaged genome to cell surface: Transcriptome 55. Murray IA, Stickel SK, Roberts RJ (2010) Sequence-specific cleavage of RNA by Type II changes during bacterial cell death triggered by loss of a restriction-modification restriction enzymes. Nucleic Acids Res 38:8257–8268. gene complex. Nucleic Acids Res 37:3021–3031. 56. Lagerbäck P, Andersson E, Malmberg C, Carlson K (2009) Bacteriophage T4 27. Keatch SA, Leonard PG, Ladbury JE, Dryden DTF (2005) StpA protein from Escherichia endonuclease II, a promiscuous GIY-YIG , binds as a tetramer to two DNA coli condenses supercoiled DNA in preference to linear DNA and protects it from substrates. Nucleic Acids Res 37:6174–6183. digestion by DNase I and EcoKI. Nucleic Acids Res 33:6540–6546. 57. Osuna R, Finkel SE, Johnson RC (1991) Identification of two functional regions in Fis: 28. Makovets S, Powell LM, Titheradge AJ, Blakely GW, Murray NE (2004) Is modification The N-terminus is required to promote Hin-mediated DNA inversion but not lambda sufficient to protect a bacterial chromosome from a resident restriction endonuclease? excision. EMBO J 10:1593–1603. Mol Microbiol 51:135–147. 58. Joseph N, Sawarkar R, Rao DN (2004) DNA mismatch correction in Haemophilus 29. Dillon SC, Dorman CJ (2010) Bacterial nucleoid-associated proteins, nucleoid structure influenzae: Characterization of MutL, MutH and their interaction. DNA Repair (Amst) and gene expression. Nat Rev Microbiol 8:185–195. 3:1561–1577.

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