DNA demethylation and tri-methylation of H3K4 at the TACSTD2 promoter are complementary players for TROP2 regulation in colorectal cancer cells.

TROP2 is a powerful cancer driver in colorectal cancer cells. Divergent epigenetic regulation mechanisms for the corresponding TACSTD2 gene exist such as miRNAs or DNA methylation. However, the role of TACSTD2 promoter hypermethylation in colorectal cancer has not been investigated yet. In this study, TROP2 expression strongly correlated with promoter methylation in different colorectal tumor cell lines. Treatment with 5-Azacytidine, a DNMT1 inhibitor, led to demethylation of the TACSTD2 promoter accompanied by an increase in TROP2 protein expression. TROP2 expression correlated with promoter methylation in vivo in human colon tumor tissue, thereby verifying promoter methylation as an important factor in the regulation of TROP2 expression in colorectal cancer. When performing a ChIP-Seq analysis in HCT116 and HT29 cells, we found that TACSTD2 promoter demethylation was accompanied by tri-methylation of H3K4. In silico analysis of GSE156613 data set confirmed that a higher binding of histone mark H3K4me3 around the TACSTD2 promoter was found in TACSTD2 high expressing tumors of colon cancer patients compared to the corresponding adjacent tumor tissue. Moreover, the link between TROP2 and the H3K4me3 code was even evident in tumors showing high intratumoral heterogeneity for TROP2 staining. Our data provide novel evidence for promoter demethylation and simultaneous gains of the active histone mark H3K4me3 across CpG-rich sequences, both being complementary mechanisms in the transcriptional regulation of TACSTD2 in colon cancer. The functional consequences of TROP2 loss in colorectal cancer needs to be further investigated.

Spectral characterization of flash and high flux x-ray radiographic sources with a magnetic Compton spectrometer.

In this work, we present a new analysis method applied to revitalize permanent magnet Compton spectrometers used to measure photon energy spectra in the MeV range. The inversion of the measured electron distribution to determine the original photon distribution is achieved via a method of consistent coupled radiation transport and magnetic field mapping of the input photon spectra to the measured electron distribution. The method of linear least squares was used to perform the unfolding of the electron distribution to the initial photon spectra, without any assumptions made regarding the electron distribution. We present an application of this method to data from a nominal 19.4 MeV flash radiographic source (the first axis of the Dual Axis Radiographic Hydro-Test Facility) capable of generating 500 R @ 1 m in ∼60 ns and a medical therapy source (a Scanditronix M22, Microtron) capable of variable energies with nominal endpoints of 6, 10, 15, and 20 MeV and an output of ∼1000-2000 R/min @ 1 m. The results provide agreement between the modeled and unfolded experimentally measured photon spectra as quantified by statistical tests, from 1.5 to 20 MeV. Experimental results are presented as well as a discussion of the novel MCNP6-based simulations and methods for reconstruction of the spectra.

Alterations in the mechanical, chemical and biocompatibility properties of low-cost polyethylene and polyester meshes after steam sterilization.

INTRODUCTION: In Africa and other Low Resource Settings (LRS), the guideline-based and thus in most cases mesh-based treatment of inguinal hernias is only feasible to a very limited extent. This has led to an increased use of low cost meshes (LCMs, mostly mosquito meshes) for patients in LRS. Most of the LCMs used are made of polyethylene or polyester, which must be sterilized before use. The aim of our investigations was to determine changes in the biocompatibility of fibroblasts as well as mechanical and chemical properties of LCMs after steam sterilization. MATERIAL AND METHODS: Two large-pored LCMs made of polyester and polyethylene in a size of 11 x 6 cm were cut and steam sterilized at 100, 121 and 134 °C. These probes and non-sterile meshes were then subjected to mechanical tensile tests in vertical and horizontal tension, chemical analyses and biocompatibility tests with human fibroblasts. All meshes were examined by stereomicroscopy, scanning electron microscopy (SEM), LDH (cytotoxicity) measurement, viability testing, pH, lactate and glycolysis determination. RESULTS: Even macroscopically, polyethylene LCMs showed massive shrinkage after steam sterilization, especially at 121 and 134 °C. While polyester meshes showed no significant changes after sterilization with regard to deformation and damage as well as tensile force and stiffness, only the unsterile polyethylene mesh and the mesh sterilized at 100 °C could be tested mechanically due to the shrinkage of the other specimen. For these meshes the tensile forces were about four times higher than for polyester LCMs. Chemical analysis showed that the typical melting point of polyester LCMs was between 254 and 269 °C. Contrary to the specifications, the polyethylene LCM did not consist of low-density polyethylene, but rather high-density polyethylene and therefore had a melting point of 137 °C, so that the marked shrinkage described above occurred. Stereomicroscopy confirmed the shrinkage of polyethylene LCMs already after sterilization at 100 °C in contrast to polyester LCMs. Surprisingly, cytotoxicity (LDH measurement) was lowest for both non-sterile LCMs, while polyethylene LCMs sterilized at 100 and 121 °C in particular showed a significant increase in cytotoxicity 48 hours after incubation with fibroblasts. Glucose metabolism showed no significant changes between sterile and non-sterile polyethylene and polyester LCMs. CONCLUSION: The process of steam sterilization significantly alters mechanical and structural properties of synthetic hernia mesh implants. Our findings do not support a use of low-cost meshes because of their unpredictable properties after steam sterilization.

An in vitro study on the biocompatibility of fibroblasts in sterile and non-sterile low-cost and commercial meshes.

INTRODUCTION: Despite several successful studies with low-cost meshes (LCM) for the treatment of inguinal hernias in India and Africa, a nationwide application has not been possible for a variety of reasons. One problem is the special preparation and sterilization of these meshes-naturally, they should comply with international standards and demands, which is often difficult to achieve in Africa. Our primary approach was to determine whether there are differences in the biocompatibility of fibroblasts between non-sterile and sterile LCMs and commercial meshes (CM). MATERIALS AND METHODS: Two polyester CMs with different pore size and a polyester LCM were examined as both sterile and non-sterile. LCM was plasma sterilized at 60 °C and steam sterilized at 134 °C. Sterile and non-sterile meshes were soaked with an antibiotic (penicillin/streptomycin) and antimycotic solution (amphotericin B). Human fibroblasts from healthy subcutaneous tissue were used. Various tests for evaluating the growth behavior and cell morphology of human fibroblasts were conducted. Semiquantitative (light microscopy) and qualitative (scanning electron microscopy) analyses were performed after 1 week and again after 12 weeks. The metabolism of fibroblasts was checked by pH measurements and glucose analyses. Biocompatibility of fibroblasts on sterile and non-sterile meshes was carried out by luminescence methods (cell viability and apoptosis) as well as calorimetric methods for proliferation determination (BrDU assay) and cytotoxicity (LDH assay). RESULTS: Light and electron microscopy revealed a moderate growth of fibroblasts on all investigated mesh types. The results of glycolysis and the pH value were within the normal range for all sterile and non-sterile meshes. In biocompatibility studies, no elevated level of apoptosis was detected. The viability measurement of mitochondrial activity of fibroblasts showed a 50% inhibition of mitochondria in all nets, with the exception of non-sterile CM, whereas mitochondrial activity was increased in the non-sterile CM. A proliferation measurement (BrdU test) revealed different growth inhibition in the sterile and non-sterile meshes. This growth inhibition was significantly stronger, particularly for non-sterile CM light meshes, than it was for the non-sterile LCM. CONCLUSION: Again, our studies show no significant differences in biocompatibility of fibroblasts between expensive and low-cost meshes. In addition, we detected fibroblast growth even in sterile meshes, independent of the mesh group. To our knowledge, the present study is the first of its kind in terms of qualitative equivalence of sterile and non-sterile in vitro mesh samples. We do not wish to create future patient studies with non-sterilized meshes saturated with antibiotics/antimycotics. However, perhaps we can prove in future studies that under semi-sterile conditions with certain LCMs, wound infection rates can be acceptable.

The relation between local structural distortion and the low-temperature magnetic anomaly in Fe7S8.

Structural defects on an atomic level can crucially impact the magnetic properties of a material. We study this phenomenon by means of magnetometry and powder neutron diffraction on a stoichiometric, monoclinic pyrrhotite (Fe7S8), which is a classic omission structure with a magnetic anomaly at about 30 K. The initial structural distortion of the pyrrhotite at 300 K caused by the vacancy arrangement decreases upon cooling, and simultaneous to the magnetic anomaly the anisotropic contraction of the unit cell homogenizes the covalency of the Fe-Fe bonds with lengths less than 3.0 Å and the Fe-S-Fe bond angles. These changes on the atomic level affect the spin-orbit coupling and the super-exchange interactions in Fe7S8, and trigger the low-temperature magnetic anomaly within a crystallographically stable system.

Identification of tetrahydrocarbazoles as novel multifactorial drug candidates for treatment of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative brain disorder and the most frequent cause of dementia. To date, there are only a few approved drugs for AD, which show little or no effect on disease progression. Impaired intracellular calcium homeostasis is believed to occur early in the cascade of events leading to AD. Here, we examined the possibility of normalizing the disrupted calcium homeostasis in the endoplasmic reticulum (ER) store as an innovative approach for AD drug discovery. High-throughput screening of a small-molecule compound library led to the identification of tetrahydrocarbazoles, a novel multifactorial class of compounds that can normalize the impaired ER calcium homeostasis. We found that the tetrahydrocarbazole lead structure, first, dampens the enhanced calcium release from ER in HEK293 cells expressing familial Alzheimer's disease (FAD)-linked presenilin 1 mutations. Second, the lead structure also improves mitochondrial function, measured by increased mitochondrial membrane potential. Third, the same lead structure also attenuates the production of amyloid-beta (Aß) peptides by decreasing the cleavage of amyloid precursor protein (APP) by ß-secretase, without notably affecting α- and γ-secretase cleavage activities. Considering the beneficial effects of tetrahydrocarbazoles addressing three key pathological aspects of AD, these compounds hold promise for the development of potentially effective AD drug candidates.

Interaction-induced partitioning and magnetization jumps in the mixed-spin oxide FeTiO3-Fe2O3.

In this study we report on jumps in the magnetic moment of the hemo-ilmenite solid solution (x)FeTiO(3)-(1-x)Fe(2)O(3) above Fe(III) percolation at low temperature (T<3 K). The first jumps appear at 2.5 K, one at each side of the magnetization loop, and their number increases with decreasing temperature and reaches 5 at T=0.5 K. The jumps occur after field reversal from a saturated state and are symmetrical in the trigger field and intensity with respect to the field axis. Moreover, an increase of the sample temperature by 2.8% at T=2.0 K indicates the energy released after the ignition of the magnetization jump, as the spin-currents generated by the event are dissipated in the lattice. The magnetization jumps are further investigated by Monte Carlo simulations, which show that these effects are a result of magnetic interaction-induced partitioning on a sublattice level.

The expression and function of NKG2D molecule on intrahepatic CD8+ T cells in chronic viral hepatitis.

The natural killer (NK) cell receptor, NKG2D is a member of the c-type lectin-activating receptor family. It is expressed by all NK cells and by a sub-population of CD8+ T cells. NKG2D engagement with its ligands directly activates NK cells and acts as a co-stimulator on CD8+ T cells. Recent reports, however, have demonstrated a role for NKG2D in direct T-cell activation in chronic inflammation. The aim of this study was to investigate the pattern of expression and the functional role of NKG2D on circulating and intrahepatic CD8+ T cells in chronic viral hepatitis. Peripheral blood lymphocytes and intrahepatic lymphocytes from 45 patients with chronic viral hepatitis (HBV and HCV) were studied. Phenotypic NKG2D expression and its functional ability to activate intrahepatic and circulating lymphocytes were analysed. Intrahepatic CD8+ T cells display increased NKG2D expression in chronic viral hepatitis in comparison with circulating CD8+ T cells. NKG2D co-stimulates intrahepatic CD8+ T cells and hepatitis B virus-specific CD8+ T cells. However, we could not demonstrate an ability to directly activate CD8+ T cells through the NKG2D signalling pathway alone. NKG2D is up-regulated on intrahepatic CD8+ T cells in type B and C chronic viral hepatitis; however, its function appears to be restricted to that of a co-stimulatory molecule.

Peripheral blood dendritic cells are phenotypically and functionally intact in chronic hepatitis B virus (HBV) infection.

Persistence of hepatitis B virus (HBV) infection is associated with reduced anti-viral T cell responses. Impaired dendritic cell (DC) function was suggested as the cause of reduced T cell stimulation in chronic HBV carriers. Thus, we compared myeloid (mDC) and plasmacytoid DC (pDC) from chronic HBV carriers and controls. Frequency and phenotype of isolated DC were analysed by fluorescence activated cell sorter staining, DC function by mixed lymphocyte reaction, cytokine bead array, intracellular cytokine staining, enzyme-linked immunosorbent assay and enzyme-linked immunospot. Expression of HBV DNA and mRNA was studied by polymerase chain reaction (PCR). Circulating total DC, mDC or pDC were not reduced in chronic HBV carriers. Isolated mDC and pDC from chronic HBV carriers exhibited similar expression of co-stimulatory molecules and alloreactive T helper cell stimulation as control DC, whether tested directly ex vivo or after in vitro maturation. Secretion of pro- and anti-inflammatory cytokines by CD40 or Toll-like receptor ligand-stimulated patient DC was intact, as was human leucocyte antigen A2-restricted HBV-specific cytotoxic lymphocyte stimulation. Although both DC populations contained viral DNA, viral mRNA was undetectable by reverse transcription-PCR, arguing against viral replication in DC. We found no quantitative, phenotypic or functional impairment of mDC or pDC in chronic hepatitis B, whether studied ex vivo or after in vitro maturation.

RNA polymerase sigma factor that blocks morphological differentiation by Streptomyces coelicolor.

The filamentous bacterium Streptomyces coelicolor undergoes a complicated process of morphological differentiation that begins with the formation of an aerial mycelium and culminates in sporulation. Genes required for the initiation of aerial mycelium formation have been termed bld (bald), describing the smooth, undifferentiated colonies of mutant strains. By using an insertional mutagenesis protocol that relies on in vitro transposition, we have isolated a bld mutant harboring an insertion in a previously uncharacterized gene, SCE59.12c, renamed here rsuA. The insertion mutant exhibited no measurable growth defect but failed to produce an aerial mycelium and showed a significant delay in the production of the polyketide antibiotic actinorhodin. The rsuA gene encodes an apparent anti-sigma factor and is located immediately downstream of SCE59.13c, renamed here sigU, whose product is inferred to be a member of the extracytoplasmic function subfamily of RNA polymerase sigma factors. The absence of rsuA in a strain that contained sigU caused a block in development, and the overexpression of sigU in an otherwise wild-type strain caused a delay in aerial mycelium formation. However, a strain in which both rsuA and sigU had been deleted was able to undergo morphological differentiation normally. We conclude that the rsuA-encoded anti-sigma factor is responsible for antagonizing the function of the sigma factor encoded by sigU. We also conclude that the sigU-encoded sigma factor is not normally required for development but that its uncontrolled activity obstructs morphological differentiation at an early stage.

Minimum detectable level of Salmonellae using a binomial-based bacterial ice nucleation detection assay (BIND).

A modified bacterial ice nucleation detection (BIND) assay was used for rapid and sensitive detection of several Salmonella species. For the BIND assay, Salmonella cells are infected with bacteriophage genetically modified to contain DNA encoding an ice nucleation protein (INP). After infection, de novo protein synthesis occurs and INPs are incorporated into the outer membrane of the organism. After supercooling (-9.3 degrees C), only buffer solutions containing transfected salmonellae freeze, causing a phase-sensitive dye to change color. This technique, and a probability-based protocol modification, provided quantitative detection with a minimum detectable level (MDL) of 2.0 +/- 0.3 S. enteritidis cells/mL in buffer (about 3 h). The MDLs for S. typhimurium DT104 and S. abaetetuba were 4.2 +/- 0.2 and 11.1 +/- 0.4 cells/mL, respectively. Using salmonellae-specific immunomagnetic bead separation technology in conjunction with the modified BIND protocol, we achieved an MDL of about 4.5 S. enteritidis cells/mL with an apparent capture efficiency of 56%.

Genomewide insertional mutagenesis in Streptomyces coelicolor reveals additional genes involved in morphological differentiation.

The filamentous soil bacterium Streptomyces coelicolor undergoes a complex cycle of morphological differentiation involving the formation of an aerial mycelium and the production of pigmented antibiotics. We have developed a procedure for generating insertional mutants of S. coelicolor based on in vitro transposition of a plasmid library of cloned S. coelicolor DNAs. The insertionally mutated library was introduced into S. coelicolor, and transposon insertions were recovered at widely scattered locations around the chromosome. Many of the insertions revealed previously uncharacterized genes, and several caused novel mutant phenotypes, such as altered pigment production, enhanced antibiotic sensitivity, delayed or impaired formation of aerial hyphae, and a block in spore formation. The sporulation mutant harbored an insertion in one of three adjacent genes that are apparently unique to Streptomyces but are each represented by at least 20 paralogs at dispersed locations in the chromosome. Individual members of the three families often are found grouped together in a characteristic arrangement, suggesting that they have a common function.

Large degree of racemization observed in the amide bond forming reaction on silica gel.

A systematic study of racemization from the coupling of DNB-L-Leu and 3-aminopropyl silica gel with several amide coupling reagents was investigated. Significant amounts of racemization were observed from all except one coupling reagent. In comparison, similar reactions completed in homogeneous solution can be accomplished with much lower racemization and in much higher yields.

Assembly line enzymology by multimodular nonribosomal peptide synthetases: the thioesterase domain of E. coli EntF catalyzes both elongation and cyclolactonization.

BACKGROUND: EntF is a 142 kDa four domain (condensation-adenylation-peptidyl carrier protein-thioesterase) nonribosomal peptide synthetase (NRPS) enzyme that assembles the Escherichia coli N-acyl-serine trilactone siderophore enterobactin from serine, dihydroxybenzoate (DHB) and ATP with three other enzymes (EntB, EntD and EntE). To assess how EntF forms three ester linkages and cyclotrimerizes the covalent acyl enzyme DHB-Ser-S-PCP (peptidyl carrier protein) intermediate, we mutated residues of the proposed catalytic Ser-His-Asp triad of the thioesterase (TE) domain. RESULTS: The Ser1138-->Cys mutant (kcat decreased 1000-fold compared with wild-type EntF) releases both enterobactin (75%) and linear (DHB-Ser)2 dimer (25%) as products. The His 1271-->Ala mutant (kcat decreased 10,000-fold compared with wild-type EntF) releases only enterobactin, but accumulates both DHB-Ser-O-TE and (DHB-Ser)2-O-TE acyl enzyme intermediates. Electrospray ionization and Fourier transform mass spectrometry of proteolytic digests were used to analyze the intermediates. CONCLUSIONS: These results establish that the TE domain of EntF is both a cyclotrimerizing lactone synthetase and an elongation catalyst for ester-bond formation between covalently tethered DHB-Ser moieties, a new function for chain-termination TE domains found at the carboxyl termini of multimodular NRPSs and polyketide synthases.

Lysine biosynthesis in Saccharomyces cerevisiae: mechanism of alpha-aminoadipate reductase (Lys2) involves posttranslational phosphopantetheinylation by Lys5.

A key step in fungal biosynthesis of lysine, enzymatic reduction of alpha-aminoadipate at C6 to the semialdehyde, requires two gene products in Saccharomyces cerevisiae, Lys2 and Lys5. Here, we show that the 31-kDa Lys5 is a specific posttranslational modification catalyst, using coenzyme A (CoASH) as a cosubstrate to phosphopantetheinylate Ser880 of the 155-kDa Lys2 and activate it for catalysis. Lys2 was subcloned from S. cerevisiae and expressed in and purified from Escherichia coli as a full-length 155-kDa enzyme, as a 105-kDa adenylation/peptidyl carrier protein (A/PCP) fragment (residues 1-924), and as a 14-kDa PCP fragment (residues 809-924). The apo-PCP fragment was covalently modified to phosphopantetheinylated holo-PCP by pure Lys5 and CoASH with a Km of 1 microM and kcat of 3 min-1 for both the PCP and CoASH substrates. The adenylation domain of the A/PCP fragment activated S-carboxymethyl-L-cysteine (kcat/Km = 840 mM-1 min-1) at 16% the efficiency of L-alpha-aminoadipate in [32P]PPi/ATP exchange assays. The holo form of the A/PCP 105-kDa fragment of Lys2 covalently aminoacylated itself with [35S]S-carboxymethyl-L-cysteine. Addition of NADPH discharged the covalent acyl-S-PCP Lys2, consistent with a reductive cleavage of the acyl-S-enzyme intermediate. These results identify the Lys5/Lys2 pair as a two-component system in which Lys5 covalently primes Lys2, allowing alpha-aminoadipate reductase activity by holo-Lys2 with catalytic cycles of autoaminoacylation and reductive cleavage. This is a novel mechanism for a fungal enzyme essential for amino acid metabolism.

The enzymological basis for resistance of herpesvirus DNA polymerase mutants to acyclovir: relationship to the structure of alpha-like DNA polymerases.

Acyclovir (ACV), like many antiviral drugs, is a nucleoside analog. In vitro, ACV triphosphate inhibits herpesvirus DNA polymerase by means of binding, incorporation into primer/template, and dead-end complex formation in the presence of the next deoxynucleoside triphosphate. However, it is not known whether this mechanism operates in vivo. To address this and other questions, we analyzed eight mutant polymerases encoded by drug-resistant viruses, each altered in a region conserved among alpha-like DNA polymerases. We measured Km and kcat values for dGTP and ACV triphosphate incorporation and Ki values of ACV triphosphate for dGTP incorporation for each mutant. Certain mutants showed increased Km values for ACV triphosphate incorporation, suggesting a defect in inhibitor binding. Other mutants showed reduced kcat values for ACV triphosphate incorporation, suggesting a defect in incorporation of inhibitor into DNA, while the rest of the mutants exhibited both altered km and kcat values. In most cases, the fold increase in Ki of ACV triphosphate for dGTP incorporation relative to wild-type polymerase was similar to fold resistance conferred by the mutation in vivo; however, one mutation conferred a much greater increase in resistance than in Ki. The effects of mutations on enzyme kinetics could be explained by using a model of an alpha-like DNA polymerase active site bound to primer/template and inhibitor. The results have implications for mechanisms of action and resistance of antiviral nucleoside analogs in vivo, in particular for the importance of incorporation into DNA and for the functional roles of conserved regions of polymerases.

Iron acquisition in plague: modular logic in enzymatic biogenesis of yersiniabactin by Yersinia pestis.

BACKGROUND: Virulence in the pathogenic bacterium Yersinia pestis, causative agent of bubonic plague, has been correlated with the biosynthesis and transport of an iron-chelating siderophore, yersiniabactin, which is induced under iron-starvation conditions. Initial DNA sequencing suggested that this system is highly conserved among the pathogenic Yersinia. Yersiniabactin contains a phenolic group and three five-membered thiazole heterocycles that serve as iron ligands. RESULTS: The entire Y. pestis yersiniabactin region has been sequenced. Sequence analysis of yersiniabactin biosynthetic regions (irp2-ybtE and ybtS) reveals a strategy for siderophore production using a mixed polyketide synthase/nonribosomal peptide synthetase complex formed between HMWP1 and HMWP2 (encoded by irp1 and irp2). The complex contains 16 domains, five of them variants of phosphopantetheine-modified peptidyl carrier protein or acyl carrier protein domains. HMWP1 and HMWP2 also contain methyltransferase and heterocyclization domains. Mutating ybtS revealed that this gene encodes a protein essential for yersiniabactin synthesis. CONCLUSIONS: The HMWP1 and HMWP2 domain organization suggests that the yersiniabactin siderophore is assembled in a modular fashion, in which a series of covalent intermediates are passed from the amino terminus of HMWP2 to the carboxyl terminus of HMWP1. Biosynthetic labeling studies indicate that the three yersiniabactin methyl moieties are donated by S-adenosylmethionine and that the linker between the thiazoline and thiazolidine rings is derived from malonyl-CoA. The salicylate moiety is probably synthesized using the aromatic amino-acid biosynthetic pathway, the final step of which converts chorismate to salicylate. YbtS might be necessary for converting chorismate to salicylate.

The nonribosomal peptide synthetase HMWP2 forms a thiazoline ring during biogenesis of yersiniabactin, an iron-chelating virulence factor of Yersinia pestis.

Pathogenic Yersinia species have been shown to synthesize a siderophore molecule, yersiniabactin, as a virulence factor during iron starvation. Here we provide the first biochemical evidence for the role of the Yersinia pestis high molecular weight protein 2 (HMWP2), a nonribosomal peptide synthetase homologue, and YbtE in the initiation of yersiniabactin biosynthesis. YbtE catalyzes the adenylation of salicylate and the transfer of this activated salicyl group to the N-terminal aryl carrier protein domain (ArCP; residues 1-100) of HMWP2. A fragment of HMWP2, residues 1-1491, can adenylate cysteine and with the resulting cysteinyl-AMP autoaminoacylate the peptidyl carrier protein domain (PCP1; residues 1383-1491) either in cis or in trans. Catalytic release of hydroxyphenylthiazoline carboxylic acid (HPT-COOH) and/or N-(hydroxyphenylthiazolinylcarbonyl)cysteine (HPT-cys) is observed upon incubation of YbtE, HMWP2 1-1491, L-cysteine, salicylate, and ATP. These products presumably arise from nucleophilic attack by water or cysteine of a stoichiometric hydroxyphenylthiazolinylcarbonyl-S-PCP1-HMWP2 intermediate. Detection of the heterocyclization capacity of HMWP2 1-1491 implies salicyl-transferring and thiazoline-forming activity for the HMWP2 condensation domain (residues 101-544) and is the first demonstration of such heterocyclization ability in a nonribosomal peptide synthetase enzyme.