From the Bacterial peptide synthetases have two common
features that appear to be strictly conserved. 1) The enzyme subunits
are co-regulated at both transcriptional and translational level. 2)
The organization of the different enzymatic domains constituting the
enzyme fulfills the "colinearity rule" according to which the order
of the domains along the chromosome parallels their functional
hierarchy. Considering the high degree of conservation of these
features, one would expect that mutations such as transcription
uncoupling and domain dissociations, deletions, duplications, and
reshuffling would result in profound effects on the quality and
quantity of synthesized peptides. To start testing this hypothesis, we
designed two mutants. In one mutant, the operon structure of surfactin
synthetase was destroyed, thus altering the concerted expression of the
enzyme subunits. In the other mutant, the thioesterase domain naturally
fused to the last amino acid binding domain of surfactin was physically dissociated and independently expressed. When the lipopeptides secreted
by the mutant Bacillus subtilis strains were purified and
characterized, they appeared to be expressed approximately at the same
level of the wild type surfactin and to be identical to it, indicating
that specific domain-domain interactions rather than coordinated
transcription and translation play the major role in determining
the correct assembly and activity of peptide synthetases.
A number of peptides with highly variable structures and a broad
range of biological activities are produced in bacteria and fungi as
secondary metabolites via a nonribosomal mechanism. The synthesis
involves large multienzyme complexes called peptide synthetases that
are organized in structural domains and utilize the thiotemplate
mechanism first described by Lipmann (1) and recently revised (2).
According to this mechanism, each domain recognizes a specific amino
acid that, after activation to the corresponding acyladenylate derivative, is covalently bound via a thioester bond to the
phosphopantetheine cofactor present on each domain. The growth of the
polypeptide chain occurs through a series of thioester bond cleavages
and simultaneous formation of amide bonds. At the end of the synthesis, the peptide is supposed to be released from the enzyme by a
thioesterase activity localized within the synthetase complex.
All prokaryotic peptide synthetases share highly conserved features (3,
4). First of all, they are organized in protein subunits consisting of
one or more amino acid binding domains. At the DNA level, the order
with which these domains are aligned along the chromosome parallels the
sequence of the amino acids in the peptide (colinearity rule). For
example, if the peptide has the sequence ABC, the domain recognizing
amino acid A will be first on the chromosome, followed by the domains
binding amino acids B and C. Secondly, the protein subunits coding
genes are located within operon-type structures, with their expression
coordinated at transcriptional level. Finally, fused at the C-terminal
end of the last amino acid binding domain, there is a thioesterase-like domain, the function of which is most probably involved in the release
of the peptide from the enzyme.
Although the striking conservation of these structural features would
suggest their crucial role in enzyme assembly and functioning, no
direct evidence of such a role has been reported so far.
In the present communication, we address two main questions. 1) Is
transcription coordination strictly required for proper enzyme assembly
and activity? 2) Can the thioesterase domain be physically dissociated
from the last amino acid binding domain without impairing peptide
synthesis?
To answer these questions we used surfactin synthetase as a model
system. This enzyme, produced by Bacillus subtilis (5, 6),
is responsible for the synthesis of the seven-amino acid lipopeptide
surfactin and is organized in three protein subunits, the first two (
srfAORF1 and srfAORF2) carrying three amino acid binding domains each
and the third subunit (srfAORF3) having a single amino acid binding
domain fused to a thioesterase moiety (TE)1 (7).
We constructed two B. subtilis mutants in which the
surfactin synthetase was modified at chromosomal level. In the first
mutant, a constitutive promoter was positioned upstream from srfAORF3, encoding the third enzyme subunit, thus making its synthesis
transcriptionally dissociated from the other two subunits, which are in
turn transcribed from the native, growth-regulated, srf
promoter (8-10). In the second construct, the DNA region encoding the
thioesterase domain was separated from the upstream region of the
leucine binding domain of srfAORF3 and expressed independently by
adding appropriate regulatory elements. The data presented here
demonstrate that the coordinate transcription of enzyme subunits and
the physical linkage between amino acid binding and thioesterase
modules are not strictly required for the correct assembly of a
functional surfactin synthetase.
To our knowledge, this is the first report describing the feasibility
of independent expression of modules and/or subunits in bacterial
peptide synthetases. These results might have important implications in
the engineering of peptide synthetases.
Enzymes and Reagents--
Restriction enzymes were from N. E. Biolabs (Schwalbach/Taunus, Germany) and were used according to the
manufacturer's instructions. For polymerase chain reaction
amplifications, the recombinant Tth DNA polymerase XL from Perkin-Elmer
was used after the protocol recommended by the manufacturer. All
chemical reagents were purchased from Sigma. Deoxyoligonucleotides were
synthesized using the DNA synthesizer ABI 394 and the reagents from
Applied Biosystem.
Plasmids and Strains--
The Escherichia coli B. subtilis shuttle vector pSM214G, a
derivative of pSM214 (11), was used for
cloning the fragments amplified from the surfactin synthetase locus of
B. subtilis JH642+ strain (7, 12). E. coli XL1
blue strain (Stratagene) was used as the host for intermediate cloning
experiments and for plasmid amplification. The suicide plasmid pDIA5304
(13) was used for the construction of the surfactin synthetase mutants of B. subtilis JH642+. Transformation into E. coli and B. subtilis were carried out as already
described (14).
Bacterial Culture Conditions--
LB agar plates supplemented
with 5% sheep blood and 5 µg/ml chloramphenicol were used for colony
isolation and rapid identification of surfactant-producing B. subtilis colonies. Typically, bacterial cultures were grown at
37 °C in VY liquid medium (25 g/liter Difco veal infusion broth, 5 g/liter yeast extract). For surfactin production, Landy medium was used
(20 g/liter glucose, 5 g/liter glutamate, 0.5 g/liter
MgSO4, 0.5 g/liter KCl, 1 g/liter
KH2PO4, 0.15 g/liter FeSO4·7H2O, 5 mg/liter MnSO4,
0.16 mg/liter CuSO4·5H2O, 0.1% yeast extract), and cells were grown for 24 h at 37 °C.
Construction of Mutant Strains--
The chromosomal regions
spanning position 21327 to 21639 (fragment I) and from position 24496 to 24790 (fragment II) of the srf locus (7) were amplified
using the following oligodeoxynucleotides: srfA-FOR
(5'-GGGGTACCCCGGTTTGTGCTTTCCTGCTCC-3'), srfA-REV
(5'-AACTGCAGCCCTTCCTGCA-TCGGCGATAGG-3'), TE-FOR
(5'-CGGAATTCATGGAAGAAACAATCGCACAAAT-3') and TE-REV
(5'-CCCCGGGCCATAGCCCAGAACCGGCGG-3'). The amplification
products were purified on MicroSpin S-400 high resolution columns
(Amersham Pharmacia Biotech), digested with the appropriate restriction
enzymes, and finally inserted into plasmid pSM214 as
KpnI-PstI and EcoRI-XmaI
fragments, respectively, after further purification on agarose gel
using the Quiaex II gel extraction kit (Qiagen). Fragment I was excised
from pSM214 by either XbaI-PstI
(promoter-carrying fragment) or KpnI-PstI (promoterless fragment) digestions and inserted into the suicide, integrative plasmid pDIA5304, generating plasmids pDIA- Analysis of Surfactin Synthetase Expression--
Total cell
proteins were analyzed on SDS-polyacrylamide gel electrophoresis
according to Laemmli (15). Briefly, cells from 2-ml culture samples
were harvested by centrifugation and lysed by resuspending them in 50 µl of 100 mM Tris-HCl, pH 6.8, 2 mg/ml lysozyme. After
incubation at 37 °C for 15 min, an equal volume of gel loading
buffer (62.5 mM Tris-HCl, pH 6.8, 5% (v/v)
2-mercaptoethanol, 4% SDS, 10% (v/v) glycerol, 0.025% (w/v)
bromphenol blue) was added, and 20 µl of each sample was loaded onto
8% SDS-polyacrylamide after a 10-min heating at 100 °C. Protein
separation was carried out at 25 mA for 3 h.
Department of Molecular Biology,
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
srfA3 and
pDIA-srfA3, respectively. Anal-ogously, fragment II was isolated from pSM214 by double digestion with either XbaI and
XmaI or with EcoRI and XmaI, and the
two fragments were cloned in pDIA5304, obtaining the plasmids
pDIA-
TE and pDIA-TE, respectively. Mutants were isolated by
transforming B. subtilis JH642+ with the four suicide
plasmids and selecting for chlor-amphenicol-resistant colonies. The
correctness of plasmid integrations was verified by polymerase chain
reaction using two oligos hybridizing in the chloramphenicol resistance
gene (CmI, 5'-ACAATAGCGACGGAGAGTTAGGTTATTGGG-3' and CmII,
5'-GCCAGTCA-TTAGGCCTATCTGACAATTCC-3') and three oligos hybridizing in
the srfA region at positions 24459-24477 (S3FOR, 5'-TGATCAGGATCAGCTGGCGGAA GAATGG-3'), 24908-24881 (S3REV:
5'-TCCGGCTGCAGCTTCTGGATCAAATCCG-3') and 20853-20873 (SRFAFOR:
5'-AGATGTTCATCAGCCATATGG-3') (see Fig. 1).
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RESULTS |
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Construction of srfA Mutants-- Two pairs of constructs in the surfactin synthetase locus were generated by using the strategy shown in Fig. 1 and described in detail under "Experimental Procedures." In the first pair, srfAORF3 was separated from the rest of the srfA operon by inserting a suicide plasmid at the end of srfAORF2. In the second pair, the suicide plasmid insertion occurred at the end of the leucine binding domain of srfAORF3, thus physically dissociating the thioesterase domain from the rest of the subunit. The constructs of each pair differ for the presence or absence of a constitutive promoter in the suicide plasmid used for integration. Therefore, the integration of the promoter-containing plasmid led to the expression of the srfA region located downstream from the integration site (in one case the region is the entire srfAORF3; in the other the region is the thioesterase domain of srfAORF3), whereas the promoterless plasmid prevented the expression of the same regions.
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Analysis of Surfactin Synthetase Expression--
The three
surfactin synthetase subunits, srfAORF1, srfAORF2, and srfAORF3, are
readily visible by Coomassie Blue staining after separation on
SDS-polyacrylamide gels of the total cell proteins from B. subtilis JH642+ (16). Therefore, the expression of the surfactin
synthetase subunits in the four srfA mutants can be followed by
analyzing the soluble proteins from bacterial cultures grown in rich
liquid medium. As shown in Fig.
3A, the three subunits were
clearly visible when total proteins from a 24-hour culture of
srfA::pDIAsrfA3 were analyzed. On the contrary and as
expected, srfAORF3 was absent in srfA::pDIAsrfA3 strain. Furthermore, in agreement with the fact that in
srfA::pDIAsrfA3 srfAORF3 transcription is under the control
of a constitutive promoter, the expression of this subunit could be
detected at the very beginning of the bacterial growth when the other
two subunits were still not visible on SDS-polyacrylamide gel
electrophoresis (Fig. 3B).
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Analysis of the Lipopeptide Produced by the srfA Mutants-- When the four srfA mutants were grown on blood agar plates, the colonies of srfA::pDIAsrfA3 and srfA::pDIA-TE strains were surrounded by typical lysis halos, indicating that a surfactin-like lipopeptide was secreted. The other two mutants, both obtained with the promoterless suicide plasmid, did not produce halos around their colonies. To quantify the amount of lipopeptide produced by srfA::pDIAsrfA3 and srfA::pDIA-TE, the surfactant was acid-precipitated from the culture supernatants of the two strains as well as from the supernatant of the wild type B. subtilis JH642+ and analyzed by TLC. Since we found that the strain carrying the srfA::pDIA-TE integration grew very poorly in liquid minimal medium, for the quantitative analysis of surfactin production, a two-medium growth was used in which the three strains were first cultured in VY-rich medium until an A600 value of 1.5 was reached, and then the cells were transferred to Landy medium, and the surfactant production analyzed 24 h later. As shown in Fig. 4, srfA::pDIAsrfA3 produced approximately the same amount of surfactant as the wild type strain, whereas in srfA::pDIA-TE, the surfactant was approximately 50% less than the wild type. The fact that the surfactants had the same RF value on TLC strongly suggested that the three strains produced identical products.
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DISCUSSION |
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The genetic organization, synthesis, and assembly of bacterial synthetases are schematically represented in Fig. 6A. As shown in this figure, the amino acid binding domains, generally located in more than one subunit and aligned along the chromosome according to the colinearity rule, are tightly coordinated at both the transcriptional and translational level. The highly conserved overall genetic and structural organization could be indicative of the relevant role this organization has in the proper synthesis and assembly of the enzymatic complex.
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For example, the colinearity rule, according to which the order of the amino acid binding domains parallels the order of the amino acids in the peptide, might lead to the assumption that by simply changing the order of the domains, different peptides can be generated. This assumption is further sustained by the strong homology existing at the amino acid level among different domains (3, 7, 17, 18).
Furthermore, the coordinated transcription and translation found in bacterial peptide synthetases could suggest that the stoichiometry among subunits needs to be finely regulated. In fact, an excess of a particular subunit in a multisubunit enzyme not only could be energetically disadvantageous but, and more importantly, could result in an incorrect interaction among subunits with the consequent generation of undesired peptides.
With the present work, we started to question to what extent this conserved organization of peptide synthetases is truly indispensable for proper enzyme synthesis and assembly or rather represents the result of peculiar mechanisms of duplication, insertion, and translocation, through which these interesting enzymes have evolved (19, 20).
In particular, we asked ourselves what might happen to the peptide synthesis if 1) subunit transcription is driven by different, independently regulated promoters and, 2) enzymatic domains located on the same protein subunit are physically dissociated and independently expressed (Fig. 6B).
Our mutant strain, pDIA-srfA3, where the srfA operon was interrupted after the ORF2, thus leading ORF3 to be expressed constitutively and independently, was still able to produce wild type surfactin. This demonstrates that the coexpression and coregulation of the different subunits of the surfactin synthetase complex are not a prerequisite for the production of an active enzyme. Moreover, it would suggest that the order by which the amino acid binding domains are aligned at the DNA level is not strictly required for the correct assembly of the multienzyme complex. In fact, since ORF3 appears in pDIA-srfA3 cells well before the other two synthetase subunits (Fig. 3B) and is synthesized about 5 kilobase pairs apart from the rest of the surfactin synthetase operon, the subunit would have equal chances to interact with either ORF1 or ORF2, unless structural constraints exist. The fact that only wild type surfactin could be purified from the supernatant of pDIA-srfA3 and the fact that its productivity was comparable to the wild type strain would suggest that such structural constraints play the most relevant role in the correct assembly of the enzyme.
Obviously, a more direct demonstration that the colinear organization of domains in peptide synthetases is not essential for enzyme assembly would be to change the order of domain coding sequences. Such experiments are currently in progress.
The data presented here also suggest that the functional domains of peptide synthetase subunits can be physically dissociated without impairing their subsequent assembly and their concerted activities. Our mutant pDIA-TE, in which the TE domain was separated from the leucine binding domain and expressed under the control of a constitutive promoter, was still able to synthesize surfactin.
Again, as in the case of pDIA-ORF3 mutant, surfactin was the only peptide formed in the supernatant of pDIA-TE culture, indicating that TE cannot proficiently interact with any other amino acid binding domain apart from the leucine binding domain of ORF3.
Therefore, specific protein-protein interactions rather than the ordered position and the progressive expression of the enzyme domains/subunits dictate the way in which peptide synthetases correctly assemble.
This might appear to contradict our recently published results on the engineering of surfactin synthetase in which, when the TE domain was moved next to either the fourth or the fifth domain of the enzyme, functional recombinant peptide synthetases were obtained that led to the synthesis of a four-amino acid and a five-amino acid lipopeptide, respectively (21).
However, in these experiments, for the thioesterase to release the four- and five-amino acid peptides, the C-terminal end of the seventh amino acid binding domain going from the pantheteine cofactor binding motif to the beginning of the thioesterase (102 amino acids) had to be included in the thioesterase sequence. This C-terminal end replaced the corresponding regions of the fourth and fifth domains in the reported surfactin synthetase fusions.
Interestingly, it has been recently reported that the thioesterase-like domain found at the end of the six-module polyketide synthetase DEBS of Saccharopolyspora erythraea can be repositioned next to the second module generating a recombinant enzyme that synthesizes a short chain polyketide (22). Also in these experiments, the grafting of the thioesterase domain was carried out maintaining the C-terminal region of the preceding acyl carrier protein domain to which it is naturally fused.
Taken together these data lead us to hypothesize that for the thioesterase to function, a specific protein-protein interaction with the preceding amino acid binding domain must be established. The region of the amino acid binding domain involved in the interaction with the thioesterase maps within the sequence spanning from the cofactor binding motif (amino acid position 1015) to the residue next to thioesterase domain (amino acid position 1117). This interaction specificity prevents the thioesterase from binding to other amino acid binding domains when the enzyme is physically separated from the domain to which it is naturally fused.
In all bacterial peptide synthetases so far characterized, why is TE constantly found fused to the C-terminal end of the last amino acid binding domain even though not strictly required for enzyme assembly and activity? The most probable explanation is provided by our finding showing that pDIA-TE mutant in which TE has been dissociated from the last leucine binding domain of surfactin synthetase is severely impaired in growth. This could indicate that when freed from the enzyme, TE can interfere with other important enzymatic functions involved in cell metabolism. The detrimental effect of free TE is supported by our failure in cloning and expressing TE both in E. coli and B. subtilis using multicopy plasmids (data not shown).
The fact that the specific interaction among domains rather than genetic organization guides the correct assembly of peptide synthetases has emerged from a number of recent observations. First of all, Marahiel and co-workers (23), in their publication describing the successful engineering of surfactin synthetase, showed that functional enzymes could be obtained when the internal core region of the leucine binding domain was replaced with the corresponding regions of other amino acid binding domains. The sequences flanking the core region, most likely involved in domain-domain interactions, were rigorously kept unchanged in these experiments. Secondly, our attempts to obtain shorter surfactin-like lipopeptides by deleting whole amino acid binding domains have mostly failed so far.2
Third, we have recently completed the characterization of the syringomycin synthetase coding region, which in Pseudomonas syringae is responsible for the synthesis of the nine-amino acid-long plant virulence factor syringomycin (24). The analysis of the organization of syringomycin synthetase has shown that this enzyme is composed of two subunits, one carrying eight amino acid binding domains and one having a single domain that recognizes the last amino acid of the peptide. The single domain subunit is encoded upstream from the eight-domain subunit, and the two coding genes are expressed under the control of different promoters.3
Our findings deliver two messages to those interested in peptide synthetase engineering. From one side, for the engineered peptide synthetase to function, reconstitution of operon-type structures and/or fused domains do not appear to be required. This may facilitate the manipulation of these enzymes, especially in consideration of the fact that large pieces of DNA generally need to be inserted in highly conserved, repeated regions. On the other hand, particular attention has to be paid to the regions involved in domain-domain interactions. Structural perturbation of these domain regions should most likely result in the failure of enzyme complex formation. The recent elucidation of the 3D structure of the phenylalanine binding domain of gramicidin synthetase (25) will be of great help in defining the regions involved in amino acid specificity and in elucidating the structural features of domain interaction.
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ACKNOWLEDGEMENTS |
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We also thank Enzo Scarlato and Rino Rappuoli for their critical reading of the manuscript. The expert secretarial assistance of Antonietta Maiorino is greatly acknowledged.
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FOOTNOTES |
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* The work was financed in part by a grant from the European Community, IV Framework Program (to G. Grandi) and in part by the contribution of the "Istituto Pasteur Fondazione Cenci Bolognetti" and the Italian Ministry of University and Scientific and Technological Research (to I. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Chiron S.p.A., Via
Fiorentina, 1 53100 Siena, Italy. Tel.: 39 577 243506; Fax: 39 577 243564; E-mail: grandi{at}iris02.biocine.it.
1 The abbreviations used are: TE, thioesterase-like domain; HPLC, high pressure liquid chromatography; ORF, open reading frame.
2 G. Grandi, unpublished observations.
3 E. Guenzi, G. Galli, and G. Grandi, manuscript in preparation.
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REFERENCES |
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