The recent increase in bacterial resistance to antibiotics has promoted the exploration of novel antibacterial materials. As a result, many researchers are undertaking work to identify new lantibiotics because of their potent antimicrobial activities. The objective of this study was to provide details of a lantibiotic-like gene cluster in Paenibacillus elgii B69 and to produce the antibacterial substances coded by this gene cluster based on culture screening.
Analysis of the P. elgii B69 genome sequence revealed the presence of a lantibiotic-like gene cluster composed of five open reading frames (elgT1, elgC, elgT2, elgB, and elgA). Screening of culture extracts for active substances possessing the predicted properties of the encoded product led to the isolation of four novel peptides (elgicins AI, AII, B, and C) with a broad inhibitory spectrum. The molecular weights of these peptides were 4536, 4593, 4706, and 4820 Da, respectively. The N-terminal sequence of elgicin B was Leu-Gly-Asp-Tyr, which corresponded to the partial sequence of the peptide ElgA encoded by elgA. Edman degradation suggested that the product elgicin B is derived from ElgA. By correlating the results of electrospray ionization-mass spectrometry analyses of elgicins AI, AII, and C, these peptides are deduced to have originated from the same precursor, ElgA.
A novel lantibiotic-like gene cluster was shown to be present in P. elgii B69. Four new lantibiotics with a broad inhibitory spectrum were isolated, and these appear to be promising antibacterial agents.
Here, we report the draft genome sequence of Paenibacillus elgiiB69, which was isolated from soil and has broad-spectrum antimicrobial activity. As far as we know, the P. elgiigenome is the largest of the Paenibacillusgenus for which genome sequences are available. Multiple sets of genes related to antibiotic biosynthetic pathways have been found in the genome.
Massetolide A is a cyclic lipopeptide (CLP) antibiotic produced by various Pseudomonas strains from diverse environments. Cloning, sequencing, site-directed mutagenesis, and complementation showed that massetolide A biosynthesis in P. fluorescens SS101 is governed by three nonribosomal peptide synthetase (NRPS) genes, designated massA, massB, and massC, spanning approximately 30 kb. Prediction of the nature and configuration of the amino acids by in silico analysis of adenylation and condensation domains of the NRPSs was consistent with the chemically determined structure of the peptide moiety of massetolide A. Structural analysis of massetolide A derivatives produced by SS101 indicated that most of the variations in the peptide moiety occur at amino acid positions 4 and 9. Regions flanking the mass genes contained several genes found in other Pseudomonas CLP biosynthesis clusters, which encode LuxR-type transcriptional regulators, ABC transporters, and an RND-like outer membrane protein. In contrast to most Pseudomonas CLP gene clusters known to date, the mass genes are not physically linked but are organized in two separate clusters, with massA disconnected from massB and massC. Quantitative real-time PCR analysis indicated that transcription of massC is strongly reduced when massB is mutated, suggesting that these two genes function in an operon, whereas transcription of massA is independent of massBC and vice versa. Massetolide A is produced in the early exponential growth phase, and biosynthesis appears not to be regulated by N-acylhomoserine lactone-based quorum sensing. Massetolide A production is essential in swarming motility of P. fluorescens SS101 and plays an important role in biofilm formation.
Pasteurella haemolytica serotype 1 is the bacterium most commonly associated with bovine shipping fever. The presence of antibodies against P. haemolytica outer membrane proteins (OMPs) correlates statistically with resistance to experimental P. haemolytica challenge in cattle. Until now, specific P. haemolytica OMPs which elicit antibodies that function in host defense mechanisms have not been identified. In this study, we have cloned and sequenced the gene encoding one such protein, PlpE. Analysis of the deduced amino acid sequence revealed that PlpE is a lipoprotein and that it is similar to an Actinobacillus pleuropneumoniae lipoprotein, OmlA. Affinity-purified, anti-PlpE antibodies recognize a protein in all serotypes of P. haemolytica except serotype 11. We found that intact P. haemolytica and recombinant E. coli expressing PlpE are capable of absorbing anti-PlpE antibodies from bovine immune serum, indicating that PlpE is surface exposed in P. haemolytica and assumes a similar surface-exposed conformation in E. coli. In complement-mediated killing assays, we observed a significant reduction in killing of P. haemolytica when bovine immune serum that was depleted of anti-PlpE antibodies was used as the source of antibody. Our data suggest that PlpE is surface exposed and immunogenic in cattle and that antibodies against PlpE contribute to host defense against P. haemolytica.
The mannopeptimycins are a novel class of lipoglycopeptide antibiotics active against multidrug-resistant pathogens with potential as clinically useful antibacterials. This report is the first to describe the biosynthesis of this novel class of mannosylated lipoglycopeptides. Included here are the cloning, sequencing, annotation, and manipulation of the mannopeptimycin biosynthetic gene cluster from Streptomyces hygroscopicus NRRL 30439. Encoded by genes within the mannopeptimycin biosynthetic gene cluster are enzymes responsible for the generation of the hexapeptide core (nonribosomal peptide synthetases [NRPS]) and tailoring reactions (mannosylation, isovalerylation, hydroxylation, and methylation). The NRPS system is noncanonical in that it has six modules utilizing only five amino acid-specific adenylation domains and it lacks a prototypical NRPS macrocyclizing thioesterase domain. Analysis of the mannopeptimycin gene cluster and its engineering has elucidated the mannopeptimycin biosynthetic pathway and provides the framework to make new and improved mannopeptimycins biosynthetically.
The environmental strain Bacillus amyloliquefaciens FZB42 promotes plant growth and suppresses plant pathogenic organisms present in the rhizosphere. We sampled sequenced the genome of FZB42 and identified 2,947 genes with >50% identity on the amino acid level to the corresponding genes of Bacillus subtilis 168. Six large gene clusters encoding nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) occupied 7.5% of the whole genome. Two of the PKS and one of the NRPS encoding gene clusters were unique insertions in the FZB42 genome and are not present in B. subtilis 168. Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis revealed expression of the antibiotic lipopeptide products surfactin, fengycin, and bacillomycin D. The fengycin (fen) and the surfactin (srf) operons were organized and located as in B. subtilis 168. A large 37.2-kb antibiotic DNA island containing the bmy gene cluster was attributed to the biosynthesis of bacillomycin D. The bmy island was found inserted close to the fen operon. The responsibility of the bmy, fen, and srf gene clusters for the production of the corresponding secondary metabolites was demonstrated by cassette mutagenesis, which led to the loss of the ability to produce these peptides. Although these single mutants still largely retained their ability to control fungal spread, a double mutant lacking both bacillomycin D and fengycin was heavily impaired in its ability to inhibit growth of phytopathogenic fungi, suggesting that both lipopeptides act in a synergistic manner.
Paenibacillus polymyxa (formerly Bacillus polymyxa) PKB1 has been identified as a potential agent for biocontrol of blackleg disease of canola, caused by the pathogenic fungus Leptosphaeria maculans. The factors presumed to contribute to disease suppression by strain PKB1 include the production of fusaricidin-type antifungal metabolites that appear around the onset of bacterial sporulation. The fusaricidins are a family of lipopeptide antibiotics consisting of a β-hydroxy fatty acid linked to a cyclic hexapeptide. Using a reverse genetic approach based on conserved motifs of nonribosomal peptide synthetases, a DNA fragment that appears to encode the first two modules of the putative fusaricidin synthetase (fusA) was isolated from PKB1. To confirm the involvement of fusA in production of fusaricidins, a modified PCR targeting mutagenesis protocol was developed to create a fusA mutation in PKB1. A DNA fragment internal to fusA was replaced by a gene disruption cassette containing two antibiotic resistance genes for independent selection of apramycin resistance in Escherichia coli and chloramphenicol resistance in P. polymyxa. Inclusion of an oriT site in the disruption cassette allowed efficient transfer of the inactivated fusA allele to P. polymyxa by intergeneric conjugation. Targeted disruption of fusA led to the complete loss of antifungal activity against L. maculans, suggesting that fusA plays an essential role in the nonribosomal synthesis of fusaricidins.
Nonribosomal peptide synthetases (NRPSs) are multimodular enzymes, found in fungi and bacteria, which biosynthesize peptides without the aid of ribosomes. Although their metabolite products have been the subject of intense investigation due to their life-saving roles as medicinals and injurious roles as mycotoxins and virulence factors, little is known of the phylogenetic relationships of the corresponding NRPSs or whether they can be ranked into subgroups of common function. We identified genes (NPS) encoding NRPS and NRPS-like proteins in 38 fungal genomes and undertook phylogenomic analyses in order to identify fungal NRPS subfamilies, assess taxonomic distribution, evaluate levels of conservation across subfamilies, and address mechanisms of evolution of multimodular NRPSs. We also characterized relationships of fungal NRPSs, a representative sampling of bacterial NRPSs, and related adenylating enzymes, including α-aminoadipate reductases (AARs) involved in lysine biosynthesis in fungi.
Phylogenomic analysis identified nine major subfamilies of fungal NRPSs which fell into two main groups: one corresponds to NPS genes encoding primarily mono/bi-modular enzymes which grouped with bacterial NRPSs and the other includes genes encoding primarily multimodular and exclusively fungal NRPSs. AARs shared a closer phylogenetic relationship to NRPSs than to other acyl-adenylating enzymes. Phylogenetic analyses and taxonomic distribution suggest that several mono/bi-modular subfamilies arose either prior to, or early in, the evolution of fungi, while two multimodular groups appear restricted to and expanded in fungi. The older mono/bi-modular subfamilies show conserved domain architectures suggestive of functional conservation, while multimodular NRPSs, particularly those unique to euascomycetes, show a diversity of architectures and of genetic mechanisms generating this diversity.
This work is the first to characterize subfamilies of fungal NRPSs. Our analyses suggest that mono/bi-modular NRPSs have more ancient origins and more conserved domain architectures than most multimodular NRPSs. It also demonstrates that the α-aminoadipate reductases involved in lysine biosynthesis in fungi are closely related to mono/bi-modular NRPSs. Several groups of mono/bi-modular NRPS metabolites are predicted to play more pivotal roles in cellular metabolism than products of multimodular NRPSs. In contrast, multimodular subfamilies of NRPSs are of more recent origin, are restricted to fungi, show less stable domain architectures, and biosynthesize metabolites which perform more niche-specific functions than mono/bi-modular NRPS products. The euascomycete-only NRPS subfamily, in particular, shows evidence for extensive gain and loss of domains suggestive of the contribution of domain duplication and loss in responding to niche-specific pressures.
The biosynthesis of many natural products of clinical interest involves large, multi-domain enzymes called nonribosomal peptide synthetases (NRPSs). In bacteria, many of the gene clusters coding for NRPSs also code for a member of the MbtH-like protein superfamily, which are small proteins of unknown function. Using MbtH-like proteins from three separate NRPS systems, we show that these proteins co-purify together with the NRPSs and influence amino acid activation. As a consequence, MbtH-like proteins are integral components of NRPSs.
Polyketide synthase (PKSs) and nonribosomal peptide synthetase (NRPSs) are large multimodular enzymes involved in biosynthesis of polyketide and peptide toxins produced by fungi. Furthermore, hybrid enzymes, in which a reducing PKS region is fused to a single NRPS module, are also responsible of the synthesis of peptide-polyketide metabolites in fungi. The genes encoding for PKSs and NRPSs have been exposed to complex evolutionary mechanisms, which have determined the great number and diversity of metabolites. In this study, we considered the most important polyketide and peptide mycotoxins and, for the first time, a phylogenetic analysis of both PKSs and NRPSs involved in their biosynthesis was assessed using two domains for each enzyme: β-ketosynthase (KS) and acyl-transferase (AT) for PKSs; adenylation (A) and condensation (C) for NRPSs. The analysis of both KS and AT domains confirmed the differentiation of the three classes of highly, partially and non-reducing PKSs. Hybrid PKS-NRPSs involved in mycotoxins biosynthesis grouped together in the phylogenetic trees of all the domains analyzed. For most mycotoxins, the corresponding biosynthetic enzymes from distinct fungal species grouped together, except for PKS and NRPS involved in ochratoxin A biosynthesis, for which an unlike process of evolution could be hypothesized in different species.
polyketide synthase; non-ribosomal peptide synthetase; phylogenies; β-ketosynthase; acyl-transferase; adenylation; condensation; secondary metabolites
The rhizosphere isolate Pseudomonas putida BW11M1 produces a mixture of cyclic lipopeptide congeners, designated xantholysins. Properties of the major compound xantholysin A, shared with several other Pseudomonas lipopeptides, include antifungal activity and toxicity to Gram-positive bacteria, a supportive role in biofilm formation, and facilitation of surface colonization through swarming. Atypical is the lipopeptide’s capacity to inhibit some Gram-negative bacteria, including several xanthomonads. The lipotetradecadepsipeptides are assembled by XtlA, XtlB and XtlC, three co-linearly operating non-ribosomal peptide synthetases (NRPSs) displaying similarity in modular architecture with the entolysin-producing enzymes of the entomopathogenic Pseudomonas entomophila L48. A shifted serine-incorporating unit in the eight-module enzyme XtlB elongating the central peptide moiety not only generates an amino acid sequence differing at several equivalent positions from entolysin, but also directs xantholysin’s macrocyclization into an octacyclic structure, distinct from the pentacyclic closure in entolysin. Relaxed fatty acid specificity during lipoinitiation by XtlA (acylation with 3-hydroxydodec-5-enoate instead of 3-hydroxydecanoate) and for incorporation of the ultimate amino acid by XtlC (valine instead of isoleucine) account for the production of the minor structural variants xantholysin C and B, respectively. Remarkably, the genetic backbones of the xantholysin and entolysin NRPS systems also bear pronounced phylogenetic similarity to those of the P. putida strains PCL1445 and RW10S2, albeit generating the seemingly structurally unrelated cyclic lipopeptides putisolvin (undecapeptide containing a cyclotetrapeptide) and WLIP (nonapeptide containing a cycloheptapeptide), respectively. This similarity includes the linked genes encoding the cognate LuxR-family regulator and tripartite export system components in addition to individual modules of the NRPS enzymes, and probably reflects a common evolutionary origin. Phylogenetic scrutiny of the modules used for selective amino acid activation by these synthetases indicates that bacteria such as pseudomonads recruit and reshuffle individual biosynthetic units and blocks thereof to engineer reorganized or novel NRPS assembly lines for diversified synthesis of lipopeptides.
The products of many bacterial non-ribosomal peptide synthetases (NRPS) are highly important secondary metabolites, including vancomycin and other antibiotics. The ability to predict substrate specificity of newly detected NRPS Adenylation (A-) domains by genome sequencing efforts is of great importance to identify and annotate new gene clusters that produce secondary metabolites. Prediction of A-domain specificity based on the sequence alone can be achieved through sequence signatures or, more accurately, through machine learning methods. We present an improved predictor, based on previous work (NRPSpredictor), that predicts A-domain specificity using Support Vector Machines on four hierarchical levels, ranging from gross physicochemical properties of an A-domain’s substrates down to single amino acid substrates. The three more general levels are predicted with an F-measure better than 0.89 and the most detailed level with an average F-measure of 0.80. We also modeled the applicability domain of our predictor to estimate for new A-domains whether they lie in the applicability domain. Finally, since there are also NRPS that play an important role in natural products chemistry of fungi, such as peptaibols and cephalosporins, we added a predictor for fungal A-domains, which predicts gross physicochemical properties with an F-measure of 0.84. The service is available at http://nrps.informatik.uni-tuebingen.de/.
Norine is the first database entirely dedicated to nonribosomal peptides (NRPs). In bacteria and fungi, in addition to the traditional ribosomal proteic biosynthesis, an alternative ribosome-independent pathway called NRP synthesis allows peptide production. It is performed by huge protein complexes called nonribosomal peptide synthetases (NRPSs). The molecules synthesized by NRPS contain a high proportion of nonproteogenic amino acids. The primary structure of these peptides is not always linear but often more complex and may contain cycles and branchings. In recent years, NRPs attracted a lot of attention because of their biological activities and pharmacological properties (antibiotic, immunosuppressor, antitumor, etc.). However, few computational resources and tools dedicated to those peptides have been available so far. Norine is focused on NRPs and contains more than 700 entries. The database is freely accessible at http://bioinfo.lifl.fr/norine/. It provides a complete computational tool for systematic study of NRPs in numerous species, and as such, should permit to obtain a better knowledge of these metabolic products and underlying biological mechanisms, and ultimately to contribute to the redesigning of natural products in order to obtain new bioactive compounds for drug discovery.
Fungal natural products containing benzodiazepinone- and quinazolinone-fused ring systems can be assembled by nonribosomal peptide synthetases (NRPS) using the conformationally restricted β-amino acid anthranilate as one of the key building blocks. We validated that the first module of the acetylaszonalenin synthetase of Neosartorya fischeri NRRL 181 activates anthranilate to anthranilyl-AMP. With this as starting point, we then used bioinformatic predictions about fungal adenylation domain selectivities to identify and confirm an anthranilate-activating module in the fumiquinazoline A producer Aspergillus fumigatus Af293 as well as a second anthranilate-activating NRPS in N. fischeri. This establishes an anthranilate adenylation domain code for fungal NRPS and should facilitate detection and cloning of gene clusters for benzodiazepine- and quinazoline-containing polycyclic alkaloids with a wide range of biological activities.
Pseudomonas species are known to be prolific producers of secondary metabolites that are synthesized wholly or in part by nonribosomal peptide synthetases. In an effort to identify additional nonribosomal peptides produced by these bacteria, a bioinformatics approach was used to “mine” the genome of Pseudomonas syringae pv. tomato DC3000 for the metabolic potential to biosynthesize previously unknown nonribosomal peptides. Herein we describe the identification of a nonribosomal peptide biosynthetic gene cluster that codes for proteins involved in the production of six structurally related linear lipopeptides. Structures for each of these lipopeptides were proposed based on amino acid analysis and mass spectrometry analyses. Mutations in this cluster resulted in the loss of swarming motility of P. syringae pv. tomato DC3000 on medium containing a low percentage of agar. This phenotype is consistent with the loss of the ability to produce a lipopeptide that functions as a biosurfactant. This work gives additional evidence that mining the genomes of microorganisms followed by metabolite and phenotypic analyses leads to the identification of previously unknown secondary metabolites.
Most filamentous ascomycete fungi produce high affinity iron chelators called siderophores, biosynthesized nonribosomally by multimodular adenylating enzymes called nonribosomal peptide synthetases (NRPSs). While genes encoding the majority of NRPSs are intermittently distributed across the fungal kingdom, those encoding ferrichrome synthetase NRPSs, responsible for biosynthesis of ferrichrome siderophores, are conserved, which offers an opportunity to trace their evolution and the genesis of their multimodular domain architecture. Furthermore, since the chemistry of many ferrichromes is known, the biochemical and structural 'rules' guiding NRPS substrate choice can be addressed using protein structural modeling and evolutionary approaches.
A search of forty-nine complete fungal genome sequences revealed that, with the exception of Schizosaccharomyces pombe, none of the yeast, chytrid, or zygomycete genomes contained a candidate ferrichrome synthetase. In contrast, all filamentous ascomycetes queried contained at least one, while presence and numbers in basidiomycetes varied. Genes encoding ferrichrome synthetases were monophyletic when analyzed with other NRPSs. Phylogenetic analyses provided support for an ancestral duplication event resulting in two main lineages. They also supported the proposed hypothesis that ferrichrome synthetases derive from an ancestral hexamodular gene, likely created by tandem duplication of complete NRPS modules. Recurrent losses of individual domains or complete modules from this ancestral gene best explain the diversity of extant domain architectures observed. Key residues and regions in the adenylation domain pocket involved in substrate choice and for binding the amino and carboxy termini of the substrate were identified.
Iron-chelating ferrichrome synthetases appear restricted to fission yeast, filamentous ascomycetes, and basidiomycetes and fall into two main lineages. Phylogenetic analyses suggest that loss of domains or modules led to evolution of iterative biosynthetic mechanisms that allow flexibility in biosynthesis of the ferrichrome product. The 10 amino acid NRPS code, proposed earlier, failed when we tried to infer substrate preference. Instead, our analyses point to several regions of the binding pocket important in substrate choice and suggest that two positions of the code are involved in substrate anchoring, not substrate choice.
The biosynthetic gene cluster for the glycopeptide-derived antitumor antibiotic zorbamycin (ZBM) was cloned by screening a cosmid library of Streptomyces flavoviridis ATCC 21892. Sequence analysis revealed 40 ORFs belonging to the ZBM biosynthetic gene cluster. However, only 23 and 22 ORFs showed striking similarities to the biosynthetic gene clusters for the bleomycins (BLMs) and tallysomycins (TLMs), respectively; the remaining ORFs do not show significant homology to ORFs from the related BLM and TLM clusters. The ZBM gene cluster consists of 16 nonribosomal peptide synthetase (NRPS) genes encoding eight complete NRPS modules, three incomplete didomain NRPS modules, and eight freestanding single NRPS domains or associated enzymes, a polyketide synthase (PKS) gene encoding one PKS module, six sugar biosynthesis genes, as well as genes encoding other biosynthesis and resistance proteins. A genetic system using Escherichia coli-Streptomyces flavoviridis intergeneric conjugation was developed to enable ZBM gene cluster boundary determinations and biosynthetic pathway manipulations.
Many bacteria use large modular enzymes for the synthesis of polyketide and peptide natural products. These multidomain enzymes contain integrated carrier domains that deliver bound substrates to multiple catalytic domains, requiring coordination of these chemical steps. Non-Ribosomal Peptide Synthetases (NRPSs) load amino acids onto carrier domains through the activity of an upstream adenylation domain. Our lab recently determined the structure of an engineered two-domain NRPS containing fused adenylation and carrier domains. This structure adopted a domain swapped dimer that illustrated the interface between these two domains. To continue our investigation, we now examine PA1221, a natural two-domain protein from Pseudomonas aeruginosa. We have determined the amino acid specificity of this new enzyme and used domain specific mutations to demonstrate that loading the downstream carrier domain within a single protein molecule occurs more quickly than loading of a non-fused carrier domain inter-molecularly. Finally, we have determined crystal structures of both the apo- and holo-PA1221 protein, the latter using a valine-adenosine vinylsulfonamide inhibitor that traps the adenylation-carrier domain interaction. The protein adopts a similar interface to that seen with the prior adenylation-carrier protein construct. A comparison of these structures with previous structures of multidomain NRPSs suggests that a large conformational change within the NRPS adenylation domains guides the carrier domain into the active site for thioester formation.
Nonribosomal peptide synthetase (NRPS) assembly lines are major avenues for the biosynthesis of a vast array of peptidyl natural products. Several hundred bacterial NRPS gene clusters contain a small (~70 residue) protein belonging to the MbtH family for which no function has been defined. Here we show that two strictly conserved Trp residues in MbtH-like proteins contribute to stimulation of amino acid adenylation in some NRPS modules. We also demonstrate that adenylation can be stimulated not only by cognate MbtH-like proteins but also by homologues from disparate natural product pathways.
Actinoplanes friuliensis produces the lipopeptide antibiotic friulimicin, which is a cyclic peptide with one exocyclic amino acid linked to a branched-chain fatty acid acyl residue. The structural relationship to daptomycin and the excellent antibacterial performance of friulimicin make the antibiotic an attractive drug candidate. The complete friulimicin biosynthetic gene cluster of 24 open reading frames from A. friuliensis was sequenced and analyzed. In addition to genes for regulation, self-resistance, and transport, the cluster contains genes encoding peptide synthetases, proteins involved in the synthesis and linkage of the fatty acid component of the antibiotic, and proteins involved in the synthesis of the nonproteinogenic amino acids pipecolinic acid, methylaspartic acid, and 2,3-diaminobutyric acid. By using heterologous gene expression in Escherichia coli, we provide biochemical evidence for the stereoselective synthesis of l-pipecolinic acid by the deduced protein of the lysine cyclodeaminase gene pip. Furthermore, we show the involvement of the dabA and dabB genes in the biosynthesis of 2,3-diaminobutyric acid by gene inactivation and subsequent feeding experiments.
Saframycin A (SFM-A), produced by Streptomyces lavendulae NRRL 11002, belongs to the tetrahydroisoquinoline family of antibiotics, and its core is structurally similar to the core of ecteinascidin 743, which is a highly potent antitumor drug isolated from a marine tunicate. In this study, the biosynthetic gene cluster for SFM-A was cloned and localized to a 62-kb contiguous DNA region. Sequence analysis revealed 30 genes that constitute the SFM-A gene cluster, encoding an unusual nonribosomal peptide synthetase (NRPS) system and tailoring enzymes and regulatory and resistance proteins. The results of substrate prediction and in vitro characterization of the adenylation specificities of this NRPS system support the hypothesis that the last module acts in an iterative manner to form a tetrapeptidyl intermediate and that the colinearity rule does not apply. Although this mechanism is different from those proposed for the SFM-A analogs SFM-Mx1 and safracin B (SAC-B), based on the high similarity of these systems, it is likely they share a common mechanism of biosynthesis as we describe here. Construction of the biosynthetic pathway of SFM-Y3, an aminated SFM-A, was achieved in the SAC-B producer (Pseudomonas fluorescens). These findings not only shed new insight on tetrahydroisoquinoline biosynthesis but also demonstrate the feasibility of engineering microorganisms to generate structurally more complex and biologically more active analogs by combinatorial biosynthesis.
The high G+C content and large genome size make the sequencing and assembly of Streptomyces genomes more difficult than for other bacteria. Many pharmaceutically important natural products are synthesized by modular polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs). The analysis of such gene clusters is difficult if the genome sequence is not of the highest quality, because clusters can be distributed over several contigs, and sequencing errors can introduce apparent frameshifts into the large PKS and NRPS proteins. An additional problem is that the modular nature of the clusters results in the presence of imperfect repeats, which may cause assembly errors. The genome sequence of Streptomyces tsukubaensis NRRL18488 was scanned for potential PKS and NRPS modular clusters. A phylogenetic approach was used to identify multiple contigs belonging to the same cluster. Four PKS clusters and six NRPS clusters were identified. Contigs containing cluster sequences were analyzed in detail by using the ClustScan program, which suggested the order and orientation of the contigs. The sequencing of the appropriate PCR products confirmed the ordering and allowed the correction of apparent frameshifts resulting from sequencing errors. The product chemistry of such correctly assembled clusters could also be predicted. The analysis of one PKS cluster showed that it should produce a bafilomycin-like compound, and reverse transcription (RT)-PCR was used to show that the cluster was transcribed.
Lipopeptide biosurfactants (LPBSs) consist of a hydrophobic fatty acid portion linked to a hydrophilic peptide chain in the molecule. With their complex and diverse structures, LPBSs exhibit various biological activities including surface activity as well as anti-cellular and anti-enzymatic activities. LPBSs are also involved in multi-cellular behaviors such as swarming motility and biofilm formation. Among the bacterial genera, Bacillus (Gram-positive) and Pseudomonas (Gram-negative) have received the most attention because they produce a wide range of effective LPBSs that are potentially useful for agricultural, chemical, food, and pharmaceutical industries. The biosynthetic mechanisms and gene regulation systems of LPBSs have been extensively analyzed over the last decade. LPBSs are generally synthesized in a ribosome-independent manner with megaenzymes called nonribosomal peptide synthetases (NRPSs). Production of active-form NRPSs requires not only transcriptional induction and translation but also post-translational modification and assemblage. The accumulated knowledge reveals the versatility and evolutionary lineage of the NRPSs system. This review provides an overview of the structural and functional diversity of LPBSs and their different biosynthetic mechanisms in Bacillus and Pseudomonas, including both typical and unique systems. Finally, successful genetic engineering of NRPSs for creating novel lipopeptides is also discussed.
lipopeptide biosurfactants (LPBSs); nonribosomal peptides; nonribosomal peptide synthetases (NRPSs)
Anguibactin, a siderophore produced by Vibrio anguillarum, is synthesized via a nonribosomal peptide synthetase (NRPS) mechanism. We have identified a gene from the V. anguillarum plasmid pJM1 that encodes a 78-kDa NRPS protein termed AngM, which is essential in the biosynthesis of anguibactin. The predicted AngM amino acid sequence shows regions of homology to the consensus sequence for the peptidyl carrier protein (PCP) and the condensation (C) domains of NRPSs, and curiously, these two domains are not associated with an adenylation (A) domain. Substitution by alanine of the serine 215 in the PCP domain and of histidine 406 in the C domain of AngM results in an anguibactin-deficient phenotype, underscoring the importance of these two domains in the function of this protein. The mutations in angM that affected anguibactin production also resulted in a dramatic attenuation of the virulence of V. anguillarum 775, highlighting the importance of this gene in the establishment of a septicemic infection in the vertebrate host. Transcription of the angM gene is initiated at an upstream transposase gene promoter that is repressed by the Fur protein in the presence of iron. Analysis of the sequence at this promoter showed that it overlaps the iron transport-biosynthesis promoter and operates in the opposite direction.
Outer membrane proteins of Pasteurella (P.) multocida have been known to be protective immunogens. Pasteurella lipoprotein E (PlpE) has been reported to be an important cross reactive outer membrane protein in P. multocida. The gene encoding the PlpE of P. multocida serotypes A: 3, B: 2 and D: 1 was amplified from the genomic DNA. The amplified products were cloned and the nucleotide sequence was determined. Sequence analysis of the recombinant clones revealed a single open reading frame of 1,011 bp, 1,008 bp and 1,017 bp encoding a protein with a calculated molecular mass of 37.829 kDa, 37.389 kDa and 37.965 kDa for serotypes A: 3, B: 2 and D: 1 respectively. The comparison of the plpE sequence in different capsular types revealed a high degree (>90%) of homology. Furthermore, the plpE gene of Haemorhhagic septicaemia causing serotype (B: 2) was expressed in E. coli and recombinant PlpE was strongly immunostained by antiserum against whole cell antigen, indicating that the protein is expressed in vivo.
Haemorhhagic septicaemia; Pasteurella multocida; PlpE