Consolidated plasmid Design for Stabilized Heterologous Production of the complex natural product Siderophore Yersiniabactin

Yersiniabactin (Ybt) is a hybrid polyketide-nonribosomal complex natural product also known as a siderophore for its iron chelation properties. The native producer of Ybt, Yersinia pestis, is a priority pathogen responsible for the plague in which the siderophore properties of Ybt are used to sequester iron and other metal species upon host infection. Alternatively, the high metal binding properties of Ybt enable a plethora of potentially valuable applications benefiting from metal remediation and/or recovery. For these applications, a surrogate production source is highly preferred relative to the pathogenic native host. In this work, we present a modification to the heterologous Escherichia coli production system established for Ybt biosynthesis. In particular, the multiple plasmids originally used to express the genetic pathway required for Ybt biosynthesis were consolidated to a single, copy-amplifiable plasmid. In so doing, plasmid stability was improved from ~30% to ≥80% while production values maintained at 20–30% of the original system, which resulted in titers of 0.5–3 mg/L from shake flask vessels.     

Role of the Yersinia pestis yersiniabactin iron acquisition system in the incidence of flea-borne plague

Plague is a flea-borne zoonosis caused by the bacterium Yersinia pestis. Y. pestis mutants lacking the yersiniabactin (Ybt) siderophore-based iron transport system are avirulent when inoculated intradermally but fully virulent when inoculated intravenously in mice. Presumably, Ybt is required to provide sufficient iron at the peripheral injection site, suggesting that Ybt would be an essential virulence factor for flea-borne plague. Here, using a flea-to-mouse transmission model, we show that a Y. pestis strain lacking the Ybt system causes fatal plague at low incidence when transmitted by fleas. Bacteriology and histology analyses revealed that a Ybt-negative strain caused only primary septicemic plague and atypical bubonic plague instead of the typical bubonic form of disease. The results provide new evidence that primary septicemic plague is a distinct clinical entity and suggest that unusual forms of plague may be caused by atypical Y. pestis strains.     

Characteristics of plague pathogen mutants,differing by pigment sorption signs

Wild-type strains of plague agent Yersinia pestis are characterized by a pigmentation phenotype (Pgm+), which includes several traits: an ability of cells to adsorb pigments (Hms+), an ability to produce siderophore yersiniabactin (Ybt+) and an ability to cause lethal infections in laboratory animals (Vir+) after subcutaneous injections. All these traits are encoded in the chromosomal pgm-locus, which gets rapidly lost due to deletion. One more trait related with the Pgm+ phenotype was detected in the present study, i.e. its siderophoric activity at 28 degrees C on the indication agar plates containing chrome azurol S (Sid+). After the four phenotypic characteristics of the Pgm+ phenotype were analyzed as well as after the four pgm-locus genes (hmsF, hmsR, irp2 and fyuA/psn) were detected by the method of hybridization and PCR, we compared 33 isogenous Pgm- mutants isolated from typical Y. pestis strain 923 by Hms-. The comparison showed that the mutants differed from each other according to the analyzed properties, which suggested that they were formed by different genetic mechanisms. Apart from the known mechanism of pgm-locus deletion, which causes an irreversible loss of Hms+, Ybt+ and Vir+ properties, two more mechanisms were detected. One of them is related with insertion damages to the pgm-locus genes, which also leads to the loss of the four traits but which can be reversed by the cultivation of cells at low temperature. The other mechanism is predetermined by unknown genetic processes ensuring the formation of mutants, which loose only their Hms+ properties and which can trigger its high-frequency reversion at 28 degrees C.     

The Yfe system of Yersinia pestis transports iron and manganese and is required for full virulence of plague

Iron acquisition in Yersinia pestis is fundamental to the success of plague pathogenesis. We have previously identified an approximately 5.6 kb region (yfe) of Y. pestis genomic DNA, capable of restoring iron-deficient growth but not siderophore production to an Escherichia coli mutant (SAB11) incapable of synthesizing the siderophore, enterobactin. The yfe locus of Y. pestis, found in both pigmented (Pgm+) and nonpigmented (Pgm-) strains, comprises five genes arranged in two distinct operons (yfeA-D and yfeE ). The larger of these, yfeABCD, encodes an ABC transport system, whose expression is iron and Fur regulated and is repressed in cells grown in the presence of manganese. Cells from a Pgm-, Yfe- (DeltayfeAB ) mutant strain of Y. pestis exhibited reduced transport of both 55Fe and 54Mn. Furthermore, cells containing an intact yfe locus showed reduced 55Fe uptake when competing amounts of MnCl2 or ZnCl2 were present, whereas 54Mn uptake was inhibited by FeCl3 but not by ZnCl2. Similarly, yfe mutants of Y. pestis exhibited growth defects on media supplemented with the iron chelators 2,2'-dipyridyl or conalbumin. These growth defects were not relieved by supplementation with MnCl2. A ybt-, DeltayfeAB mutant of Y. pestis was completely avirulent in mice infected intravenously (LD50 > 1.7 x 107 cfu) compared with its parental ybt-, yfe+ strain, which had an LD50 of < 12. In addition, compared with its ybt+, yfe+ parent, a ybt+, DeltayfeAB mutant of Y. pestis had an approximately 100-fold increase in the LD50 from a subcutaneous route of infection. These data suggest that the Yfe and Ybt systems may function effectively to accumulate iron during different stages of the infectious process of bubonic plague.     

Salicylic acid, yersiniabactin, and pyoverdin production by the model phytopathogen Pseudomonas syringae pv. tomato DC3000: synthesis, regulation, and impact on tomato and Arabidopsis host plants

A genetically tractable model plant pathosystem, Pseudomonas syringae pv. tomato DC3000 on tomato and Arabidopsis thaliana hosts, was used to investigate the role of salicylic acid (SA) and iron acquisition via siderophores in bacterial virulence. Pathogen-induced SA accumulation mediates defense in these plants, and DC3000 contains the genes required for the synthesis of SA, the SA-incorporated siderophore yersiniabactin (Ybt), and the fluorescent siderophore pyoverdin (Pvd). We found that DC3000 synthesizes SA, Ybt, and Pvd under iron-limiting conditions in culture. Synthesis of SA and Ybt by DC3000 requires pchA, an isochorismate synthase gene in the Ybt genomic cluster, and exogenous SA can restore Ybt production by the pchA mutant. Ybt was also produced by DC3000 in planta, suggesting that Ybt plays a role in DC3000 pathogenesis. However, the pchA mutant did not exhibit any growth defect or altered virulence in plants. This lack of phenotype was not attributable to plant-produced SA restoring Ybt production, as the pchA mutant grew similarly to DC3000 in an Arabidopsis SA biosynthetic mutant, and in planta Ybt was not detected in pchA-infected wild-type plants. In culture, no growth defect was observed for the pchA mutant versus DC3000 for any condition tested. Instead, enhanced growth of the pchA mutant was observed under stringent iron limitation and additional stresses. This suggests that SA and Ybt production by DC3000 is costly and that Pvd is sufficient for iron acquisition. Further exploration of the comparative synthesis and utility of Ybt versus Pvd production by DC3000 found siderophore-dependent amplification of ybt gene expression to be absent, suggesting that Ybt may play a yet unknown role in DC3000 pathogenesis.     

Yersinia ironomics: comparison of iron transporters among Yersiniapestis biotypes and its nearest neighbor, Yersinia pseudotuberculosis

Although Yersinia pestis epidemic biovars and Yersinia pseudotuberculosis are recently diverged, highly related species, they cause different diseases via disparate transmission routes. Since iron transport systems are important for iron acquisition from hosts and for survival in the environment, we have analyzed potential iron transport systems encoded by epidemic and non-epidemic or endemic strains of Y. pestis as well as two virulent Y. pseudotuberculosis strains. Computational biology analysis of these genomes showed a high degree of identity/similarity among 16 proven or possible iron/heme transporters identified. Of these, 7 systems were essentially the same in all seven genomes analyzed. The remaining 9 loci had 2–6 genetic variations among these genomes. Two untested, potential siderophore-dependent systems appear intact in Y. pseudotuberculosis but are disrupted or absent in all the endemic Y. pestis strains as well as the epidemic strains from the antiqua and mediaevalis biovars. Only one of these two loci are obviously disrupted in Y. pestis CO92 (epidemic orientalis biovar). Experimental studies failed to identify a role for hemin uptake systems in the virulence of pneumonic plague and suggest that Y. pestis CO92 does not make a siderophore other than Ybt.     

Epimerization of an L-cysteinyl to a D-cysteinyl residue during thiazoline ring formation in siderophore chain elongation by pyochelin synthetase from Pseudomonas aeruginosa

The thiazoline-containing siderophores pyochelin, yersiniabactin, and Micacocidin A all have D-thiazoline rings, participating in high-affinity chelation of ferric iron. However, studies with pyochelin (Pch) synthetase and yersiniabactin (Ybt) synthetase reconstituted from pure protein components have shown that only L-cysteine is activated and tethered as a covalent aminoacyl-S-enzyme intermediate. Nor are any of the canonical epimerase domains of nonribosomal peptide synthetase (NRPS) assembly lines found in the Ybt or Pch synthetase modules. Here, we report that the PchE subunit of the Pch synthetase exchanges solvent deuterium into the C(2) center of the thiazoline moieties during siderophore chain elongation. Both PchE and HMWP2, from Ybt synthetase, subunits have a 310-360-residue insert in their amino acid activation domains that look like defective methyltransferase (MT) domains. We suggest these inserts are noncanonical epimerase domains, reversibly deprotonating and reprotonating acyl-S-enzyme intermediates at the C(2) locus. The PchE subunit does not epimerize the Cys-S-enzyme intermediate, but once amide bond formation from a benzoyl-S-PchE donor is catalyzed by the cyclization (Cy) domain of PchE, the N-benzoyl-Cys-S-PchE intermediate is present as a D,L-mixture. The subsequent phenylthiazolinyl-S-PchE intermediate, arising from cyclodehydration of the N-benzoyl-Cys-S-PchE intermediate, is likewise a D,L-mixture on hydrolytic release and enantiomer analysis. These results suggest a default role for MT domains of NRPS assembly lines in generating alpha-carbanionic species from thioester intermediates during siderophore chain elongation.     

Molecular cloning and expression of genetic determinants for the iron uptake system mediated by the Vibrio anguillarum plasmid pJM1

Plasmid pJM1 from an invasive strain of Vibrio anguillarum encodes an iron uptake system which mediates the biosynthesis of a siderophore and a membrane receptor for the iron-siderophore complex. This system has been associated with the ability of V. anguillarum to cause hemorrhagic septicemic disease in marine fish. Recombinant derivatives containing essential regions of the pJM1-mediated iron uptake system cloned into cosmid vector pVK102 were introduced into low-virulence iron uptake-deficient V. anguillarum strains by using a trifactor mating procedure with helper plasmid pRK2013. Three recombinant clones, pJHC-T7, pJHC-T11, and pJHC-T2612, possessed genetic determinants for receptor activity. Production of receptor activity was correlated in all three cases with the presence of OM2, an 86-kilodalton outer membrane protein which was induced under iron-limiting conditions. Two of the clones, pJHC-T7 and pJHC-T2612, also coded for the production of siderophore activity, although at a much lower level than the wild type. Strains harboring either of these two clones were still unable to grow under iron-limiting conditions. This inability was overcome only when other indigenous pJM1 derivatives were present in the cells in addition to the recombinant cosmids. This restoration of high siderophore production and ability to grow under iron-limiting conditions was achieved even when the indigenous plasmids possessed lesions in genes involved in siderophore activity or in both siderophore and receptor production. Thus, another function mediated by plasmid pJM1, possibly a transacting factor, may play a role in the regulation of siderophore production. Results of experimental infections demonstrated that restoration of the ability to grow under conditions of iron limitations by introduction of an recombinant clone into one of the low-virulence V. anguillarum strains was correlated with an increase in bacterial pathogenicity.     

Chromosome-mediated iron uptake system in pathogenic strains of Vibrio anguillarum.

We describe in this work a new iron uptake system encoded by chromosomal genes in pathogenic strains of Vibrio anguillarum. This iron uptake system differs from the plasmid-encoded anguibactin-mediated system present in certain strains of V. anguillarum in several properties. The siderophore anguibactin is not utilized as an external siderophore, and although characteristic outer membrane proteins are synthesized under iron-limiting conditions, these are not related to the plasmid-mediated outer membrane protein OM2 associated with ferric anguibactin transport. Furthermore, the siderophore produced by the plasmidless strains may be functionally related to enterobactin as demonstrated by bioassays with enterobactin-deficient mutants, although its behavior under various chemical treatments suggested major differences from that siderophore. Hybridization experiments suggested that the V. anguillarum chromosome-mediated iron uptake system is unrelated genetically to either the anguibactin or enterobactin-associated iron assimilation systems.     

Increased production of yersiniabactin and an anthranilate analog through media optimization

Yersiniabactin (Ybt) is a mixed nonribosomal peptide‐polyketide natural product that binds a wide range of metals with the potential to impact processes requiring metal retrieval and removal. In this work, we substantially improved upon the heterologous production of Ybt and an associated anthranilate analog through systematic screening and optimization of culture medium components. Specifically, a Plackett‐Burman design‐of‐experiments methodology was used to screen 22 components and to determine those contributing most to siderophore production. L‐cysteine, L‐serine, glucose, and casamino acids significantly contributed to the production of both compounds. Using this approach together with metabolic engineering of the base biosynthetic process, Ybt and the anthranilate analog titers were increased to 867 ± 121 mg/L and 16.6 ± 0.3 mg/L, respectively, an increase of ～38 and ～79‐fold relative to production in M9 medium. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1193–1200, 2017     

Isolation and characterization of autoagglutination factor of Yersinia pestis Hms− cells

An approach for isolation of an autoagglutination factor (AF) from Hms(-) cells of the plague agent has been developed. Purified AF has been obtained and characterized in physicochemical properties. The AF is found to be a complex of a 17.5-kD protein with a low molecular weight peptide component, which binds iron ions and shows siderophore activity. This low molecular weight component is responsible for hydrophobic properties and immunochemical activity of the AF, as well as for its ability to interact with the plague diagnosticum L-413c bacteriophage.     

Iron uptake system medicated by Vibrio anguillarum plasmid pJM1.

Plasmid pJM1 from an invasive strain of Vibrio anguillarum mediates an iron-sequestering system that is associated with the ability of this bacterium to cause septicemia in marine fishes. This plasmid-mediated iron uptake system was analyzed by using mutations caused by transposon Tnl. Restriction endonuclease analysis of iron uptake-deficient and -proficient derivatives generated by insertion of Tnl and molecular cloning experiments permitted us to localize the plasmid regions involved in the process of iron sequestration to a stretch of about 20 kilobase pairs. In addition, the existence of two plasmid-mediated components involved in the process of iron uptake in V. anguillarum was defined: a diffusible substance which functions as a siderophore and a nondiffusible receptor for complexes of iron-siderophore, which we have tentatively identified as the pJM1 plasmid-mediated outer membrane protein OM2 of V. anguillarum.     

Identification of an ABC transporter required for iron acquisition and virulence in Mycobacterium tuberculosis

Iron availability affects the course of tuberculosis infection, and the ability to acquire this metal is known to be essential for replication of Mycobacterium tuberculosis in human macrophages. M. tuberculosis overcomes iron deficiency by producing siderophores. The relevance of siderophore synthesis for iron acquisition by M. tuberculosis has been demonstrated, but the molecules involved in iron uptake are currently unknown. We have identified two genes (irtA and irtB) encoding an ABC transporter similar to the YbtPQ system involved in iron transport in Yersinia pestis. Inactivation of the irtAB system decreases the ability of M. tuberculosis to survive iron-deficient conditions. IrtA and -B do not participate in siderophore synthesis or secretion but are required for efficient utilization of iron from Fe-carboxymycobactin, as well as replication of M. tuberculosis in human macrophages and in mouse lungs. We postulate that IrtAB is a transporter of Fe-carboxymycobactin. The irtAB genes are located in a chromosomal region previously shown to contain genes regulated by iron and the major iron regulator IdeR. Taken together, our results and previous observations made by other groups regarding two other genes in this region indicate that this gene cluster is dedicated to siderophore synthesis and transport in M. tuberculosis.     

Response of the cyanobacterium,Oscillatoria tenuis,to low iron environments: the effect on growth rate and evidence for siderophore production

Cyanobacteria vary in their ability to grow in media contaning low amounts of biologically available iron. Some strains, such as Oscillatoria tenuis, are well adapted to thrive in low-iron environments. We investigated the mechanism of iron scavenging in O. tenuis and found that this cyanobacterium has a siderophore-mediated iron transport system that differs significantly from the traditional hydroxamate-siderophore transport system reported from other cyanobacteria. Unlike other cyanobacteria, this strain produces two types of siderophores, a hydroxamate-type siderophore and a catechol-type siderophore. Production of these two siderophores is expressed at two different iron levels in the medium, suggesting two different iron regulated uptake systems. We compared the production of each siderophore with the growth rate of the culture and found that the production of the catechol siderophore enhances the growth rate of the cyanobacterium, whereas the cells maintain lower than maximal growth rates when only the hydroxamate-type siderophore is being produced.Abbreviation EDDA ethylene diamine di-(o-hydroxyphenylacetic acid)     

The ability of rifampin-resistant Escherichia coli to colonize the mouse intestine is enhanced by the presence of a plasmid-encoded aerobactin-iron(III) uptake system

Rifampin-resistant Escherichia coli are known to be poor colonizers of the animal intestine. In this report, we show that the colonizing ability of rifampin-resistant E. coli cells is increased dramatically in the presence of the aerobactin-mediated iron(III) uptake system. In contrast, the colonization by nalidixic acid-resistant E. coli does neither depend on the aerobactin-iron(III) nor on the dicitrate-iron(III) uptake system. Likewise, it does not depend on the production of the siderophore enterochelin.     

Crystal structure of ferric-yersiniabactin, a virulence factor of Yersinia pestis

Yersiniabactin (Ybt), the siderophore produced by Yersinia pestis, has been crystallized successfully in the ferric complex form and the crystal structure has been determined. The crystals are orthorhombic with a space group of P2(1)2(1)2(1) and four distinct molecules per unit cell with cell dimensions of a=11.3271(+/-0.0003)A, b=22.3556(+/-0.0006)A, and c=39.8991(+/-0.0011)A. The crystal structure of ferric Ybt shows that the ferric ion is coordinated as a 1:1 complex by three nitrogen electron pairs and three negatively charged oxygen atoms with a distorted octahedral coordination. The molecule displays a Delta absolute configuration with chiral centers at N2, C9, C10, C12, C13, and C19 in R, R, R, R, S, S configurations, respectively. Few of the crystal structures of siderophores have been solved, and those which have been are of simple hydroxamate and catechol types such as ferrioxamine B and agrobactin. To our knowledge this is the first report of the ferric crystal structure of 5-member heterocycle siderophore.