Portability of the thiolation domain in recombinant pyoverdine non-ribosomal peptide synthetases
© Calcott and Ackerley. 2015
Received: 1 March 2015
Accepted: 28 July 2015
Published: 13 August 2015
Non-ribosomal peptide synthetase (NRPS) enzymes govern the assembly of amino acids and related monomers into peptide-like natural products. A key goal of the field is to develop methods to effective recombine NRPS domains or modules, and thereby generate modified or entirely novel products. We previously showed that substitution of the condensation (C) and adenylation (A) domains in module 2 of the pyoverdine synthetase PvdD from Pseudomonas aeruginosa led to synthesis of modified pyoverdines in a minority of cases, but that more often the recombinant enzymes were non-functional. One possible explanation was that the majority of introduced C domains were unable to effectively communicate with the thiolation (T) domain immediately upstream, in the first module of PvdD.
To test this we first compared the effectiveness of C-A domain substitution relative to T-C-A domain substitution using three different paired sets of domains. Having previously demonstrated that the PvdD A/T domain interfaces are tolerant of domain substitution, we hypothesised that T-C-A domain substitution would lead to more functional recombinant enzymes, by maintaining native T/C domain interactions. Although we successfully generated two recombinant pyoverdines, having a serine or a N5-formyl-N5-hydroxyornithine residue in place of the terminal threonine of wild type pyoverdine, in neither case did the T-C-A domain substitution strategy lead to substantially higher product yield. To more comprehensively examine the abilities of non-native T domains to communicate effectively with the C domain of PvdD module 2 we then substituted the module 1 T domain with 18 different T domains sourced from other pyoverdine NRPS enzymes. In 15/18 cases the recombinant NRPS was functional, including 6/6 cases where the introduced T domain was located upstream of a C domain in its native context.
Our data indicate that T domains are generally able to interact effectively with non-native C domains, contrasting with previous findings that they are not generally portable upstream of epimerisation (E) or thioesterase (TE) domains. This offers promise for NRPS recombination efforts, but also raises the possibility that some C domains are unable to efficiently accept non-native peptides at their donor site due to steric constraints or other limitations.
The modular nature of the NRPS assembly line offers promise for the creation of rationally modified peptides, or even combinatorial libraries of novel products, by recombining NRPS domains or modules at a genetic level. The primary substrate-specifying role of the A domain has made this domain a key target for NRPS recombination experiments. However, efforts to generate novel peptide products by modifying A domain substrate specificity, or by substituting in a non-synonymous A domain (that is, one which specifies an alternative monomer to the A domain it is replacing) have generally been unsuccessful [7, 8]. Numerous in vitro e.g., [9, 10] and in vivo [11, 12] studies have demonstrated that the C domain typically “proof-reads” the downstream acceptor substrate (Fig. 1d) in a manner that prevents incorporation of non-synonymous monomers into the peptide product. Structural studies have also indicated that disruption of the native C/A domain interface during A domain substitution may further impair the activity of recombinant NRPS modules . Nonetheless, we found that five out of five synonymous A domain substitutions into PvdD (a bi-modular NRPS from Pseudomonas aeruginosa PAO1 that incorporates two L-threonine residues at the C-terminus of the peptide siderophore pyoverdine ) yielded recombinant enzymes that were highly active in vivo, whereas nine out of nine non-synonymous A domain substitutions were inactive [11, 12]. The 100 % success rate of the synonymous A domain substitutions suggests that disruption of native C/A or A/T domain interfaces was not a major factor restricting our ability to generate functional recombinant NRPS enzymes.
Results and discussion
Comparison of T-C-A and C-A domain substitution variants
To substitute alternative domains into PvdD we used an integrating vector bearing a copy of the pvdD gene that had restriction sites introduced, to enable replacement of either the native C-A domains of the second NRPS module, or the native T-C-A domains spanning the first and second modules (Fig. 2a, b). Three T-C-A domain substitution constructs were created and compared to equivalent constructs in which only the corresponding C-A domain substitutions had been made. Two of the C-A domain pairings encode a serine residue in their native context, and PvdD C-A substitution constructs bearing each of these alternative domain pairings had been analysed in our previous study ; in that work one (here called CA-Ser1) was non-functional and did not yield any detectable pigment, whereas the other (CA-Ser2) was found to incorporate a serine residue into pyoverdine. The third C-A domain pairing (used to make construct CA-fhOrn) was from a N5-formyl-N5-hydroxyornithine activating pyoverdine synthetase module from P. fluorescens SBW25, which had not previously been tested in domain substitution studies. The corresponding T-C-A domain substitution constructs were named TCA-Ser1, TCA-Ser2 and TCA-fhOrn.
Each C-A and T-C-A domain substitution construct was transformed into a pvdD mutant strain of P. aeruginosa PAO1, and analysed for production of pyoverdine (structure depicted in Fig. 3a) alongside a restriction site positive control strain (CA-Wt) and an empty plasmid negative control strain (pvdD − ). As a preliminary test for pyoverdine production, each strain was spotted onto iron-limiting King’s B agar plates in either the absence (Fig. 3b) or presence (Fig. 3c) of 200 μg.ml−1 of ethylenediamine-N,N’-bis(2-hydroxyphenylacetic acid) (EDDHA; an iron-chelating agent that prevents passive uptake of iron). Of the C-A domain substitution strains, those bearing constructs CA-Ser2 and CA-fhOrn were fluorescent under UV and able to grow in the presence of EDDHA, whereas the strain containing construct CA-Ser1 was neither fluorescent nor able to out-compete EDDHA for iron. The T-C-A domain substitution strains showed similar results to the corresponding C-A domain substitution strains; that is, the strains bearing domain substitution constructs TCA-Ser2 and TCA-fhOrn were both fluorescent and able to grow in the presence of EDDHA, whereas the strain containing construct TCA-Ser1 was not.
To more accurately quantify the levels of pyoverdine production, the absorbance (Fig. 3d) and fluorescence (Fig. 3e) for each T-C-A and C-A domain substitution strain was measured from the supernatant of 24 h cultures. As previously demonstrated, relative absorbance is a less sensitive but more linear measure of the relative amounts of pyoverdine present, whereas fluorescence is better able to detect low levels of pyoverdine . By either measure, there did not appear to be a substantial difference between the C-A and T-C-A domain substitution strategies in terms of the overall yield of pigment produced.
Analysis of T domain portability
The lack of evidence for T-C-A domain substitution being a fundamentally superior strategy to C-A domain substitution for generating functional recombinant PvdD enzymes, together with a relative paucity of suitable contiguous T-C-A domain combinations available for substitution, and technical difficulties in amplifying and cloning such large DNA inserts, dissuaded us from building additional T-C-A domain substitution constructs. Instead, to focus more specifically on whether transplanted T domains might have difficulty in communicating effectively with non-native C domains, a series of T domain substitutions were made by replacing the first T domain of PvdD according to the substitution scheme represented in Fig. 2c.
When considered in the context of altered domain interactions, T domain substitutions are fundamentally similar to C-A domain substitutions; that is, both establish a scenario in which there is perturbation of (i) native A/T domain interactions; (ii) upstream C and downstream T domain interactions; and (iii) upstream T and downstream C domain interactions (Fig. 2a, 2c). As we previously found five out of five synonymous A domain substitutions into PvdD to be active [11, 12], it can be inferred that A domains are readily able to communicate with non-native T domains. We therefore reasoned that our proposed T domain substitution strategy would shed light on whether non-native T and C domains can also communicate effectively.
Thus, only 3 out of the 18 T domain substitution strains were completely unable to synthesise pyoverdine, none of these being strains in which the native Tc domain of PvdD had been substituted by another Tc domain. These data suggest that T domains transplanted upstream of a non-native C domain generally retain functionality, contrasting with earlier studies in which TC domains placed upstream of non-native E  or TE [16–19] domains were severely impaired or inactive. Our data also confirm that the boundaries we selected between the PvdD A-T and T-C domains were appropriate recombination sites for T domain substitution. This is an important point; in one other previous study it was found that the TTE domain from the single module indigoidine synthetase BpsA from Streptomyces lavendulae was unable to substitute for the TTE domain of the closely related single module indigoidine synthetase IndC from Photorhabdus luminescens until optimal recombination sites were identified .
In previous work we found that a majority of C-A domain substitutions in the second module of PvdD yielded inactive recombinant enzymes . We hypothesised that this might stem from the T domain of the first PvdD module being unable to communicate effectively with the newly-introduced C domain. If this hypothesis were true, then T-C-A domain substitutions would be expected to prove superior to C-A domain substitutions as a means of generating novel non-ribosomal peptide products. Although T-C-A domain substitution has previously been used to successfully create a bi-modular recombinant NRPS that was functional in vitro , the authors did not construct the equivalent C-A domain substitution construct to compare relative activities. Here, while we were able to use T-C-A domain substitution to successfully generate an entirely new pyoverdine species that had fhOrn at the C-terminus, and as an alternative means of producing a serine-substituted pyoverdine we had previously generated via C-A domain substitution , we did not observe substantial differences between the T-C-A substitution and C-A domain substitution strategies in terms of product yield.
We also found that T domains were generally tolerant of being relocated immediately upstream of the C domain of the terminal PvdD module, particularly if the introduced T domain had been located immediately upstream of a C domain in its native context. That is, within their new environment they were generally capable of interacting effectively with non-native C and A domains upstream, and a non-native C domain downstream. This capability appeared to be independent of the amino acid identity shared between the introduced T domains and the PvdD T domain that they were replacing. For example, the TCt, TE and TTE domains that shared the highest sequence identity with the PvdD T domain were actually the least active when substituted into PvdD (Additional file 1: Table S1). We consider this general portability of T domains to locations upstream of C domains to be quite remarkable, in light of the extreme conformational and functional demands that are placed on T domains [13, 25–27], as well as previous studies indicating that TC domains are not generally portable upstream of E or TE domains [15–19]. From a biotechnology perspective it is a promising finding, as it suggests that T domain functional incompatibility is unlikely to be a major driver of recombinant NRPS inactivity following C-A or T-C-A domain substitution.
Collectively, our data suggest that the low success rates of our previous C-A domain substitutions into module 2 of PvdD  were not due to inability of the newly introduced C domains to communicate effectively with the PvdD module 1 T domain. Rather, we propose that the loss of function may have been due to inability of the introduced C domain to receive the large incoming pyoverdine peptide at its donor site, possibly due to steric constraints. The original work of Belshaw et al., which demonstrated no side-chain amino acid specificity at the donor site of C domains, used only single amino acids as donor substrates . In contrast, later work by Stein et al. identified specificity within the C domain donor site for some dipeptides . Similarly, Clugston et al.  observed stereo specificity at the C domain donor site towards a tetrapeptide, but not towards the single amino acid substrate. The studies by Stein et al.  and Clugston et al.  strongly suggest that the size and composition of the incoming donor peptide can influence activity. Thus, steric constraints at the donor site position might explain the variable success rate of C domain substitutions, with some C domains being more tolerant of the new peptide chain. Ultimately, this would mean that both A and C-A domain substitution strategies are impeded by C domain limitations – the former by acceptor site selectivity, and the latter by donor site constraints. Overall, a greater understanding of C domain specificity is needed as it may aid in future efforts to perform functional NRPS domain substitutions.
Bacterial strains and growth conditions
E. coli DH5α, P. aeruginosa PAO1 and P. putida KT2440 were sourced from existing Ackerley lab stocks. P. syringae pv. phaseolicola 1448A was generously provided by Prof. John Mansfield (Imperial College, London), and P. fluorescens SBW25 by Prof. Paul Rainey (Massey University, Auckland, NZ). All strains were grown in LB media with shaking at 200 rpm, at 28 °C for P. syringae pv. phaseolicola 1448A and 37 °C for other strains. For maintenance of plasmids, tetracycline was added to a final concentration of 15 μg/ml for E. coli and 100 μg/ml for P. aeruginosa PAO1. Where necessary, to prevent passive uptake of iron, EDDHA was added to a final concentration of 200 μg.ml−1.
General DNA methodology
All PCR primers and plasmids used in this study are shown in Additional file 2: Tables S2 and S3, respectively. Primers were designed to amplify pyoverdine NRPS domains from the genomes of fluorescent pseudomonads based on previous annotation by Owen and Ackerley  (P. syringae 1448a), Moon et al.  (P. fluorescens SBW25), and Ravel and Cornelis  (P. putida KT2440) as well as the NRPS analysis tools available online (http://nrps.igs.umaryland.edu/nrps/; ). PCR reactions used Phusion™ DNA polymerase (Finnzymes; Espoo, Finland). Plasmids created in this work were sequence verified by Macrogen Inc. (Seoul, South Korea).
C-A and T-C-A domain substitution
C-A and T-C-A domain substitution plasmids were constructed from pSW196  in E. coli DH5α. C-A domain constructs were created as previously described , with the primers CA-fhOrn_Fwd and CA-fhOrn_Rev used to amplify the new C-A domain pairing. To generate the T-C-A domain substitution plasmid pTCA, the C-A domains from the first module of pvdD were PCR amplified using primers CATfwd and CA-Wt_Rev and ligated into pSW196 using the NotI and SacI restriction sites. The reverse primer introduced a NotI site in exactly the same location that was used for A domain substitutions previously . Then, the T-TE domains from the second module of pvdD were introduced into the construct as previously described for the C-A domain substitution plasmid . Target T-C-A domains were then PCR amplified using the corresponding primer sets listed in Additional file 2: Table S2, and introduced into the substitution plasmids via NotI restriction digest and ligation. Correct orientation of the introduced T-C-A domain amplicons was confirmed by sequencing. The resulting plasmids were then transformed into P. aeruginosa PAO1 for single-copy integration at the attB locus .
T domain substitution
The domain substitution plasmid pST1 was derived from the plasmid pTCA. The second module of pvdD was amplified using the primers CATTE_Fwd and TTeRev. Next the PCR product and pTCA vector were each digested with NotI and SacI, and the PCR product was ligated into pTCA. This replaced the T-Te domains of pTCA with the PCR amplified second module of pvdD. The primer CATTE_Fwd had added NotI and SpeI restriction sites immediately upstream of the C domain of the second module of pvdD, which allowed the subsequent insertion of T domains into these restriction sites. T domains for substitution into pST1 were amplified from the genomes of different fluorescent pseudomonads as indicated in Fig. 5 and Additional file 1: Table S1, using the primers listed in Additional file 2: Table S2 (primers being named according to the T domain they amplify, with F indicating a forward primer, and R a reverse primer; e.g., the primers for creating strain C1 are named C1F and C1R). As per the C-A and T-C-A domain substitutions, the T domain substitution plasmids were transformed into P. aeruginosa PAO1.
Measurement of pyoverdine production
For measuring pyoverdine levels in liquid media, strains were grown in 200 μL of LB in a 96 well plate for 24 h at 37 °C. This starter culture was used to inoculate M9 media containing 0.2 % (w/v) L-arabinose and 4 g/l succinate (pH 7.0) at a 20x dilution and total volume of 200 μL. After 24 h of growth, cultures were centrifuged to pellet bacteria, and then 100 μL of supernatant transferred to a fresh 96 well plate. Supernatant was diluted 2x in fresh M9 media to give a total volume of 200 μL. Fluorescence (ex. 400 nm/ em. 440 nm) and absorbance (400 nm) were measured using an EnSpire 2300 Multilabel Reader (Perkin Elmer, Waltham, MA, USA).
Cultures were grown according to the methods for measurement of pyoverdine production, above. Cells were pelleted by centrifugation and a sample of supernatant was mixed with matrix solution (500 μL acetonitrile, 500 μL ultrapure water, 1 μL trifluoroacetic acid, 10 μg α-Cyano-4-hydroxycinnamic acid) in a volumetric sample to matrix ratio ranging from 1:5 to 1:20. Aliquots of 1 μl from each sample were spotted in duplicate onto an Opti-TOF® 384 well MALDI plate (Applied Biosystems, Foster City, CA) and allowed to dry at room temperature. Spots were analyzed using a MALDI TOF/TOF 5800 mass spectrometer (Applied Biosystems) in positive ion mode. Each spot was externally calibrated using cal2 calibration mixture (Applied Biosystems). Peaks in spectra were labelled in Data Explorer (Applied Biosystems).
Availability of supporting data
We thank Associate Professor Bill Jordan at the Victoria University of Wellington Centre for Biodiscovery for access to the Proteomics Facility; and Professor Iain Lamont at Otago University for his generous gift of the EDDHA used in this study. This work was supported by the Royal Society of New Zealand Marsden Fund [contract number VUW0901] and the Victoria University Research Fund.
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