1 School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK
2 Phico Therapeutics Ltd, Babraham Hall, Babraham, Cambridge CB2 4AT, UK
3 Department of Biological Chemistry, John Innes Centre, Norwich NR4 7UH, UK
4 Schools of Biological Sciences and Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, UK
Correspondence
Richard Bowater
r.bowater{at}uea.ac.uk
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ABSTRACT |
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These authors contributed equally to this work.
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INTRODUCTION |
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Due to the essential involvement of ligA in replication, its inactivation leads to the non-viability of most bacteria. However, several temperature-sensitive (ts) strains of E. coli were isolated during the 1970s and shown to have mutations in ligA (Dermody et al., 1979; Gellert & Bullock, 1970
; Karam et al., 1979
; Konrad et al., 1973
; Modrich & Lehman, 1971
). E. coli GR501 is one of the most ligase-deficient of these strains, and initial characterization suggested that its ts phenotype was due to a mutation in ligA, resulting in a reduction in DNA replication at high temperatures (Dermody et al., 1979
). This conclusion has been reinforced by the observation that it can be complemented by DNA ligases that participate in replication in other systems, including human DNA ligase I (Kodama et al., 1991
) and T7 DNA ligase (Doherty et al., 1996
). Although E. coli GR501 has been particularly useful for the analysis of DNA ligase function in bacteria, there has been little characterization of the nature and biochemical consequences of the mutation in this strain.
Recently, an NAD+-DNA ligase was identified within the genome of Amsacta moorei entomopoxvirus (Sriskanda et al., 2001), although functional NAD+-DNA ligases have not been characterized in eukaryotic genomes. Thus, NAD+-DNA ligases have been suggested as possible targets for broad-spectrum antibacterial compounds (Georlette et al., 2003
; Gong et al., 2004
; Kaczmarek et al., 2001
; Lee et al., 2000
; Singleton et al., 1999
; Sriskanda & Shuman, 2002
). If substantial progress is to be made in the development of inhibitors that are specific to NAD+-DNA ligases, it is vital that in vivo models are utilized to test the efficacy of such compounds. This has recently been demonstrated in elegant studies of the antibacterial nature of pyridochromanones, which are good inhibitors of the LigA from several bacteria (Brotz-Oesterhelt et al., 2003
; Gong et al., 2004
). Complementation experiments using E. coli GR501 demonstrated that these compounds do not act on human DNA ligase I, an ATP-DNA ligase (Brotz-Oesterhelt et al., 2003
). To understand the relationship of the mutation in E. coli GR501 to the structure and function of E. coli LigA, it is imperative that the molecular details of the mutation in this strain are identified. Here, we identify the mutation in the ligA gene of E. coli strain GR501, analyse its expression and overexpress the protein in a recombinant form. Biochemical analysis is used to pinpoint the molecular basis for the ts mutation in LigA from E. coli GR501.
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METHODS |
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For liquid-culture growth, single colonies from plates grown at 30 °C were inoculated into 5 ml liquid medium and grown overnight at 30 °C. These cultures were diluted 100-fold into 5 ml fresh medium and incubated at the required temperature. Growth of bacteria was detected by monitoring OD600 every 30 min for the first 2 h and subsequently every 15 min for the remainder of the incubation period.
For analysis of the viability of the various strains harbouring different plasmids, the appropriate cultures were grown in LB (with antibiotic if required) at 30 °C overnight. Viable cell counts were determined by plating 200 µl of a 106 dilution of the overnight culture onto LB agar plates and counting the colonies after aerobic incubation at 30 °C or 43 °C for 24 h.
Cloning of DNA ligases.
Genomic DNAs were prepared from appropriate overnight cultures using a Wizard Genomic DNA purification kit (Promega). Cloning of E. coli K-12 NAD+-DNA ligase (LigA; 671 amino acids) has been described previously (Wilkinson et al., 2003). LigA from E. coli GR501 (LigA251) was prepared in a similar manner: amplified from genomic DNA isolated from E. coli GR501 by PCR with a proof-reading DNA polymerase. Note that the 5' primer contained an NdeI site and that the 3' primer contained a BamHI site. PCR products were cloned using the Zero Blunt TOPO Cloning kit (Invitrogen) and sequenced to confirm that the recombinant gene was the same as that identified in E. coli GR501 genomic DNA. Fragments were excised from the TOPO vectors using the NdeI and BamHI sites and cloned into pET16b (Novagen). Proteins overexpressed from this vector contain a 10-His tag within an extra 21 amino acids (2·5 kDa) at the N-terminus.
To allow overexpression of proteins in E. coli GR501, full-length ligases plus the His-tag were excised from pET-16b vectors using the NcoI and BamHI sites and cloned into pTRC99A (Amersham Pharmacia). In control experiments analysing expression of proteins from pTRC99A, T4 DNA ligase was expressed from pRBL (Ren et al., 1997) and Im9, an inhibitor of the colicin E9, was expressed from pRJ354 (Wallis et al., 1995
).
Protein purification.
For protein expression, all E. coli cultures were grown at 37 °C in LB containing ampicillin and chloramphenicol. The pET16b derivatives were transformed into E. coli BL21 (DES) pLysS, and cells were plated on LB agar containing antibiotics and grown overnight. Single colonies were inoculated into 5 ml liquid medium, grown overnight and diluted 100-fold into fresh medium (50500 ml). After growth to mid-exponential phase (OD600=0·40·6), protein expression was induced by addition of IPTG to 0·4 mM. After 4 h further incubation, cells were harvested, sonicated and centrifuged to separate soluble and insoluble fractions. DNA ligases were purified from the soluble fraction using columns with affinity for the His-tag (HiTrap Chelating HP, Amersham Pharmacia). After concentration using Vivaspin 20 ml concentrators with a 5000 molecular weight cut-off PES membrane, the samples were loaded on to a HiLoad 16/60 Superdex 75 prep grade column. The column was run at 1 ml min1 for 2 h in 20 mM Tris, pH 7·5, 200 mM NaCl. The OD280 was measured to find the peak fractions, which were collected, pooled and concentrated. Typically, full-length DNA ligases eluted after 45 min. Protein concentrations were determined by the Bradford method (Bio-Rad Protein Assay). In general, lower levels of expression were obtained for LigA251 than for LigA: standard amounts of pure protein obtained from each litre of induced culture were 100 mg for LigA and 15 mg for LigA251. The thermal stability of LigA and LigA251 was analysed by SDS-PAGE after incubation of 2 µg (30 pmol) of protein at temperatures between 4 and 37 °C for 21 h. For long-term storage at 80 °C, glycerol was added to a final concentration of 20 % (v/v).
Analysis of ligation activity.
In vitro assays of ligation activity were performed using a double-stranded 40 bp DNA substrate carrying a single-strand nick between bases 18 and 19 (Timson & Wigley, 1999). This substrate was created in TBE buffer by annealing an 18-mer (5'-GTA AAA CGA CGG CCA GTG-3') and a 22-mer (5'-AAT TCG AGC TCG GTA CCC GGG G-3') to a complementary 40-mer (5'-CCC CGG GTA CCG AGC TCG AAT TCA CTG GCC GTC GTT TTA C-3'). At the 5' end, the 18-mer contained a fluorescein molecule and the 22-mer was phosphorylated.
The nicked 40 bp substrate was used to assay the in vitro ligation activity of each enzyme. Time-course assays used 750 pmol DNA in 50 µl reactions and different amounts of LigA and LigA251, and were performed at various temperatures and times. Reactions were conducted in the presence of 26 µM NAD+ in 30 mM Tris, pH 8·0, 4 mM MgCl2, 1 mM DTT and 50 µg BSA ml1. At the end of the incubation, 5 µl samples were mixed with an equal volume of formamide loading buffer, heated to 95 °C, loaded onto a 15 % polyacrylamide/urea gel (10x10 cm) and run at 300 V for 1 h in 1x TBE. Reaction products on the gel were visualized and quantified using a Molecular Dynamics Storm phosphorimager.
To assay for ligation activity in vivo, we tested whether different plasmid constructs could complement E. coli GR501 at temperatures that are normally restrictive to growth. Following the guidelines for the use of E. coli GR501, cells were transformed with pTRC99A containing the gene for the relevant DNA ligase and grown on LB agar containing ampicillin at 30 °C or 43 °C.
Analysis of LigA expression.
Bacterial cultures were grown at the required temperature and, at exactly OD600=0·7, 5 ml of culture was harvested and resuspended in 100 µl Tris/glycine SDS gel loading buffer (Sambrook & Russell, 2001). In some experiments, 0·4 mM IPTG was added at OD600=0·2 to induce overexpression of recombinant DNA ligases. Cell extracts were fractionated by SDS-PAGE, and LigA and LigA251 were detected by a standard protocol for Western blotting (Western Blue Express, Promega). Adenylated and deadenylated forms of LigA were resolved by electrophoresis at 100 V for 5 h on 15 % SDS-PAGE, whilst detection of His-tagged proteins was performed after electrophoresis at 150 V for 1 h on 8 % SDS-PAGE. The primary antibody to E. coli LigA (Davids Biotechnologie) was a rabbit polyclonal raised against purified LigA (containing His-tag), and the primary antibody to the His-tag was a mouse monoclonal (Amersham Pharmacia). Appropriate secondary antibodies were conjugated to alkaline phosphatase (Promega).
Sequence analysis and molecular modelling.
Sequences of DNA ligases were identified from the NCBI database (http://www.ncbi.nlm.nih.gov/). Alignment of protein sequences was performed using the CLUSTAL W method (DNAStar LaserGene MegAlign). A molecular model for E. coli LigA was generated using the X-ray crystallographic structure of Thermus filiformis NAD+-DNA ligase (Lee et al., 2000) (Protein Database accession number 1DGT). The structure was automatically generated using SWISS-MODEL (http://www.expasy.ch/spdbv/) (Guex & Peitsch, 1997
) and visualized using RasMol. Since the structure of T. filiformis DNA ligase does not contain high-resolution details of all amino acids (Lee et al., 2000
), only amino acids 1586 are contained in our molecular model of E. coli LigA.
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RESULTS |
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E. coli GR501 was obtained from the E. coli Genetic Stock Center (CGSC). To confirm that this strain had a temperature-sensitive mutation in ligA, we tested for complementation of the strain with functional DNA ligases expressed from pTRC99A, which can be used to express genes in E. coli under the control of an IPTG-inducible strong promoter (Ren et al., 1997). Previous studies have confirmed that expression of DNA ligases from this vector allows growth of E. coli GR501 at temperatures that are normally non-permissive (Brotz-Oesterhelt et al., 2003
; Kodama et al., 1991
; Ren et al., 1997
; Wilkinson et al., 2003
). Two controls were performed to confirm that complementation of growth at non-permissive temperatures was due to the expression of DNA ligases which are functional in E. coli GR501. Firstly, the thermosensitivity of cells harbouring the empty vector (pTRC99A) was assessed. Secondly, to confirm that the complementation was not due to non-specific protein expression, bacteria were transformed with pRJ345 (Wallis et al., 1995
), a pTRC99A-derived plasmid which expresses the colicin inhibitor Im9 and has no DNA ligase functions. E. coli GR501 strains harbouring these derivatives of pTRC99A were grown on plates at 30 or 43 °C. This assay was carried out without IPTG, as it had been shown that the vector expression system allowed a high level of protein synthesis even in the absence of the inducer (see below). As expected, growth was observed at 30 °C (Fig. 1
a). However, cells lacking a wild-type DNA ligase grew more slowly than cells encoding such an enzyme from the vector. In contrast, only plasmids expressing E. coli LigA and T4 DNA ligase complemented the temperature-sensitive mutation and allowed E. coli GR501 to grow well on plates at 43 °C. Note that these observations confirm that mutations in the NAD+-DNA ligase of E. coli can be complemented by overexpression of an ATP-DNA ligase (Doherty et al., 1996
; Kodama et al., 1991
).
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Since these data suggest that LigA251 is expressed at 42 °C in E. coli GR501, we assessed whether the sequence of the protein could explain the ts mutation. Genomic DNA was prepared from E. coli GR501, and sequencing of its ligA251 revealed a single base substitution, compared to ligA in E. coli K-12, of cytosine to thymine at base pair 43. This mutation results in a change from Leu to Phe at position 15 of the protein (Fig. 3). Although this residue is not absolutely conserved in all NAD+-DNA ligases, it is always a hydrophobic amino acid. In the NAD+-DNA ligases, this amino acid resides within the bi-helix that is close to the N-terminus. Since this region of LigA is important for binding of NAD+ (Sriskanda & Shuman, 2002
), the ts mutation in E. coli GR501 is likely to affect binding of NAD+ and the subsequent adenylation of LigA.
Biochemical analysis of LigA from E. coli GR501
Previously, we have purified an active recombinant form of LigA from E. coli K-12 with a 10-His-tag at the N-terminus (Wilkinson et al., 2003). Using a similar strategy, ligA251 from E. coli GR501 was amplified from genomic DNA using a proof-reading polymerase and cloned into the expression vector pET-16b. DNA sequencing confirmed that the only mutation in the cloned gene was the cytosine to thymine transition at base pair 43, which produces a change from Leu to Phe at amino acid 15 of the protein. The protein product (LigA251) was overexpressed and purified by affinity chromatography, with further purification by gel-filtration chromatography. Analysis by SDS-PAGE detected no proteins other than full-length DNA ligase (data not shown).
In vitro analysis of purified protein was used to test whether or not the ts phenotype of E. coli GR501 was due to reduced protein stability at higher temperatures. SDS-PAGE was used to analyse if there was a difference in the stability of wild-type and ts LigA when incubated at different temperatures for extended periods. Some fragmentation of proteins was observed during extended incubation (21 h) at 42 °C, but the effects were no worse for LigA251 than for LigA (Fig. 4a). Thus, in vitro analysis suggests that the mutation does not decrease the overall stability of the protein at high temperature.
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Effect of overexpression of LigA251 on E. coli GR501 viability
The above data show that LigA251 retained low levels of DNA ligation activity at 42 °C (Fig. 4). Since the non-viability of cultures at higher temperatures is likely to be due to insufficient DNA end-joining activity, we reasoned that overexpression of LigA251 might complement and allow growth of the ts strain. Following procedures outlined above for other DNA ligases, LigA251 was cloned into pTRC99A and transformed into E. coli GR501. These bacteria were able to grow well on plates lacking IPTG at 42 °C (Table 2
), indicating that overexpression of LigA251 from pTRC99A could complement the ts mutation. To confirm this observation, E. coli GR501 overexpressing either LigA or LigA251 was grown in liquid culture at various temperatures. In all cases, complementation of E. coli GR501 by expression of LigA251 or LigA resulted in similar growth patterns, as indicated at 40 and 42 °C in Fig. 5
a. Thus, overexpression of LigA251 was able to overcome the growth problems that normally result from the reduced activity of this mutated LigA. Levels of expression of the recombinant proteins at 42 °C were analysed by Western blot using a primary antibody to the His-tag (Fig. 5b
). Similar levels of expression were observed at 30 °C (data not shown). Note that significant levels of recombinant protein were detected in the absence of IPTG, indicating that expression from the strong promoter of pTRC99A was not effectively inhibited in E. coli GR501. There was some regulation at the lac-based promoter, since addition of 0·4 mM IPTG increased expression of the recombinant proteins by a further five to tenfold (Fig. 5b
). Comparison with known amounts of purified recombinant LigA allowed estimation of the expression levels of LigA251 from the chromosome of E. coli GR501 and from pTRC99A in the absence of IPTG (data not shown). This analysis identified that the amount of DNA ligase expressed from the vector pTRC99A was 20- to 50-fold higher than that expressed from the chromosomal gene.
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DISCUSSION |
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E. coli GR501 is one of the most ligase-deficient strains identified, and the ts phenotype is a consequence of problems with the completion of DNA replication at elevated temperature (Dermody et al., 1979). The mutation can be complemented by a variety of DNA ligases, indicating that the ts mutation is related to the function of E. coli ligA. Interestingly, the strain can be complemented by both NAD+- and ATP-DNA ligases (Brotz-Oesterhelt et al., 2003
; Doherty et al., 1996
; Kodama et al., 1991
; Wilkinson et al., 2003
). This reinforces the conclusion that the mutation is related to effects on replication, and supports the use of this strain in studies that aim to identify inhibitors that are specific to the NAD+-versions of the DNA ligases (Brotz-Oesterhelt et al., 2003
).
Temperature-sensitive strains contain conditional-lethal mutations: they result in lethality under restrictive conditions, but retain normal function under permissive conditions. Such ts mutations can arise via different processes, producing effects at the level of expression or activity of the ts gene at the non-permissive temperature. We have confirmed that LigA251 is expressed at similar levels at all temperatures tested. In vitro ligation assays have confirmed that the ts DNA ligase is active, but has reduced activity, at higher temperatures. Compared to wild-type LigA, LigA251 has reduced activity at all temperatures, but the difference in activity is exaggerated at temperatures that are non-permissive for growth.
These observations suggest that E. coli GR501 is temperature sensitive because its DNA ligase activity is insufficient to sustain growth at non-permissive temperatures. Our observed 60-fold difference in the rates of ligation by LigA and LigA251 at 42 °C is less than the effect of ts mutations on the DNA ligation activity of E. coli cell extracts (Dermody et al., 1979; Lehman, 1974
). These differences may indicate that another cofactor influences the ligation activity of crude extracts, or they may reflect limitations in the accuracy of the different assays of DNA ligation. Interestingly, we observed that a 20- to 50-fold overexpression of LigA251 overcomes the ts phenotype, which is in reasonable agreement with the effect of the mutation on the in vitro ligation activity of the protein at 42 °C. However, moving from 30 to 42 °C has only a small effect on the activity of LigA251, and it is unlikely that this is sufficient to explain the pronounced consequences for viability. Rather, the overall effect is likely due to a combination of factors affecting the bacteria. Although E. coli GR501 has good viability at temperatures up to 40 °C, it grows quite slowly on LB agar plates, suggesting that the growth rate of the ts strain may be compromised even at low temperatures. Switching growth to higher temperatures applies additional stresses to the bacteria, which may mean that DNA ligase has a more important role to play due to the fact that more replication forks are likely to be active, or perhaps the enzyme is required to carry out more DNA repair events. Thus, the relatively small change in biochemical activity of the mutated DNA ligase may be linked to a large physiological effect.
For E. coli GR501, the level of adenylated LigA251 appeared to be reduced at 42 °C compared to 30 °C and also in comparison to wild-type strains at the same temperature. Preliminary in vitro experiments confirmed that LigA251 was less readily adenylated by NAD+ than LigA (unpublished data). A thorough in vitro biochemical analysis is required to understand the full implications of the ts mutation on the reaction mechanism.
Sequencing of genomic and cloned DNA established that the mutation in ligA251 of E. coli GR501 is a cytosine to thymine transition at base 43, which leads to the substitution of Phe for Leu at residue 15 of the protein (LigA251). The effects of mutations in this position of E. coli LigA have not been studied before, but the N-terminal 38 residues are required for adenylation of the protein (Sriskanda et al., 1999). Amino acids in the homologous position to Leu15 are conserved as hydrophobic residues in all NAD+-DNA ligases (Fig. 3
), suggesting that it has an important function. The fact that both Leu and Phe are hydrophobic may explain why LigA251 retains some DNA ligase activity. However, the introduction of this change clearly has some effect on protein activity. To aid evaluation of the effect of this mutation on LigA function, we used the X-ray crystallographic structure of LigA from Thermus filiformis (Lee et al., 2000
) to generate a molecular model of E. coli LigA. Fig. 6
shows the backbone structure of the molecular model, with highlighting of the side-chains of Leu15 and the active site lysine (Lys115). Clearly, no direct interactions between Leu15 and the active site are predicted from this model. The main difference in the structure of Leu and Phe is the bulky benzene ring in the side chain of Phe. Although the relationship of the amino acid mutation to enzymic activity or structure has not been directly demonstrated at this time, the molecular model suggests that the alteration of amino acid 15 from Leu to Phe may alter the packing involving
-helix A, which is close to the N-terminus. This bi-helix is implicated in the binding of NAD+ to LigA (Georlette et al., 2003
; Sriskanda & Shuman, 2002
), supporting our proposal that the temperature sensitivity of LigA251 is a consequence of factors altering the rate of adenylation of the protein.
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ACKNOWLEDGEMENTS |
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Received 29 April 2004;
revised 14 July 2004;
accepted 13 August 2004.
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