Vinyl sulfonamide based thermosetting composites via thiol-Michael polymerization.

OBJECTIVE: To assess the performance of thiol Michael photocurable composites based on ester-free thiols and vinyl sulfonamides of varying monomer structures and varied filler loadings and to contrast the properties of the prototype composites with conventional BisGMA-TEGDMA methacrylate composite. METHODS: Synthetic divinyl sulfonamides and ester-free tetrafunctional thiol monomers were utilized for thiol-Michael composite development with the incorporation of thiolated microfiller. Polymerization kinetics was investigated using FTIR spectroscopy. Resin viscosities were assessed with rheometry. Water uptake properties were assessed according to standardized methods. Thermomechanical properties were analyzed by dynamic mechanical analysis. Flexural modulus/strength and flexural toughness were measured on a universal testing machine in three-point bending testing mode. RESULTS: The vinyl sulfonamide-based thiol-Michael resin formulation demonstrated a wide range of viscosities with a significant increase in the functional group conversion when compared to the BisGMA-TEGDMA system. The two different types of vinyl sulfonamide under investigation demonstrated significant differences towards the water sorption. Tertiary vinyl sulfonamide did not undergo visible swelling whereas the secondary vinyl sulfonamide composite swelled extensively in water. With the introduction of rigid monomer into the polymer matrix the glass transition temperature increased and so increased the toughness. Glassy thiol-Michael composites were obtained by ambient curing. SIGNIFICANCE: Employing the newly developed step-growth thiol-Michael resins in dental composites will provide structural uniformity, improved stability and lower water sorption.

Evaluation of biofilm formation on novel copper-catalyzed azide-alkyne cycloaddition (CuAAC)-based resins for dental restoratives.

OBJECTIVE: For the past several decades, the resins used in dental restorations have been plagued with numerous problems, including their implication in biofilm formation and secondary caries. The need for alternative resins is critical, and evaluation of biofilm formation on these resins is essential. The aim of this study was to evaluate in vitro biofilm formation on the surface of novel copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC)-based resins and composites. METHODS: CuAAC-based resins/composites made from varying azide monomers and different copper concentrations were compared with BisGMA-TEGDMA resins/composites that served as the control. Biofilms were formed using a mono-species model containing a luciferase-expressing strain of Streptococcus mutans. Luciferase activity was measured and the number of viable bacteria was enumerated on biofilms associated with each resin and composite. RESULTS: A significant reduction (p<0.05) in luciferase activity, and the number of viable bacteria recovered from biofilms on CuAAC-based resins and composites was observed in comparison to biofilms associated with the BisGMA-TEGDMA controls. SIGNIFICANCE: CuAAC-based resins do still allow for the formation of biofilms; however, the statistically significant reduction of growth that was associated with the CuAAC resin may enhance the longevity of restorations that incorporate CuAAC-based materials.

Purification and structural characterization of siderophore (corynebactin) from Corynebacterium diphtheriae.

During infection, Corynebacterium diphtheriae must compete with host iron-sequestering mechanisms for iron. C. diphtheriae can acquire iron by a siderophore-dependent iron-uptake pathway, by uptake and degradation of heme, or both. Previous studies showed that production of siderophore (corynebactin) by C. diphtheriae is repressed under high-iron growth conditions by the iron-activated diphtheria toxin repressor (DtxR) and that partially purified corynebactin fails to react in chemical assays for catecholate or hydroxamate compounds. In this study, we purified corynebactin from supernatants of low-iron cultures of the siderophore-overproducing, DtxR-negative mutant strain C. diphtheriae C7(ß) ΔdtxR by sequential anion-exchange chromatography on AG1-X2 and Source 15Q resins, followed by reverse-phase high-performance liquid chromatography (RP-HPLC) on Zorbax C8 resin. The Chrome Azurol S (CAS) chemical assay for siderophores was used to detect and measure corynebactin during purification, and the biological activity of purified corynebactin was shown by its ability to promote growth and iron uptake in siderophore-deficient mutant strains of C. diphtheriae under iron-limiting conditions. Mass spectrometry and NMR analysis demonstrated that corynebactin has a novel structure, consisting of a central lysine residue linked through its α- and ε- amino groups by amide bonds to the terminal carboxyl groups of two different citrate residues. Corynebactin from C. diphtheriae is structurally related to staphyloferrin A from Staphylococcus aureus and rhizoferrin from Rhizopus microsporus in which d-ornithine or 1,4-diaminobutane, respectively, replaces the central lysine residue that is present in corynebactin.

Alanine racemase mutants of Burkholderia pseudomallei and Burkholderia mallei and use of alanine racemase as a non-antibiotic-based selectable marker.

Burkholderia pseudomallei and Burkholderia mallei are category B select agents and must be studied under BSL3 containment in the United States. They are typically resistant to multiple antibiotics, and the antibiotics used to treat B. pseudomallei or B. mallei infections may not be used as selective agents with the corresponding Burkholderia species. Here, we investigated alanine racemase deficient mutants of B. pseudomallei and B. mallei for development of non-antibiotic-based genetic selection methods and for attenuation of virulence. The genome of B. pseudomallei K96243 has two annotated alanine racemase genes (bpsl2179 and bpss0711), and B. mallei ATCC 23344 has one (bma1575). Each of these genes encodes a functional enzyme that can complement the alanine racemase deficiency of Escherichia coli strain ALA1. Herein, we show that B. pseudomallei with in-frame deletions in both bpsl2179 and bpss0711, or B. mallei with an in-frame deletion in bma1575, requires exogenous D-alanine for growth. Introduction of bpsl2179 on a multicopy plasmid into alanine racemase deficient variants of either Burkholderia species eliminated the requirement for D-alanine. During log phase growth without D-alanine, the viable counts of alanine racemase deficient mutants of B. pseudomallei and B. mallei decreased within 2 hours by about 1000-fold and 10-fold, respectively, and no viable bacteria were present at 24 hours. We constructed several genetic tools with bpsl2179 as a selectable genetic marker, and we used them without any antibiotic selection to construct an in-frame ΔflgK mutant in the alanine racemase deficient variant of B. pseudomallei K96243. In murine peritoneal macrophages, wild type B. mallei ATCC 23344 was killed much more rapidly than wild type B. pseudomallei K96243. In addition, the alanine racemase deficient mutant of B. pseudomallei K96243 exhibited attenuation versus its isogenic parental strain with respect to growth and survival in murine peritoneal macrophages.

Development and use of a selectable, broad-host-range reporter transposon for identifying environmentally regulated promoters in bacteria.

This report describes the development and use of TnKnXSp, a selectable broad-host-range reporter transposon with a promoterless aphA gene. Insertion of TnKnXSp into the chromosome of a kanamycin-susceptible bacterium confers resistance to kanamycin only if aphA is transcribed from an active promoter adjacent to the insertion site. We designed TnKnXSp as a tool for identifying environmentally regulated promoters in bacteria and developed general methods for initial characterization of any TnKnXSp integrant. To identify putative iron-regulated promoters in Corynebacterium diphtheriae, we constructed TnKnXSp integrants and identified a subgroup that expressed kanamycin resistance under low-iron, but not high-iron, conditions. We characterized representative integrants with this phenotype, located the TnKnXSp insertion in each, and demonstrated that transcription of aphA was repressed under high-iron vs. low-iron growth conditions. We also demonstrated that TnKnXSp can be used in bacteria other than C. diphtheriae, including Escherichia coli and Bacillus subtilis. Our findings validate TnKnXSp as a useful tool for identifying environmentally regulated promoters in bacteria.

An allelic exchange system for compliant genetic manipulation of the select agents Burkholderia pseudomallei and Burkholderia mallei.

Burkholderia pseudomallei and B. mallei are Gram-negative bacterial pathogens that cause melioidosis in humans and glanders in horses, respectively. Both bacteria are classified as category B select agents in the United States. Due to strict select-agent regulations, the number of antibiotic selection markers approved for use in these bacteria is greatly limited. Approved markers for B. pseudomallei include genes encoding resistance to kanamycin (Km), gentamicin (Gm), and zeocin (Zeo); however, wild type B. pseudomallei is intrinsically resistant to these antibiotics. Selection markers for B. mallei are limited to Km and Zeo resistance genes. Additionally, there are few well developed counter-selection markers for use in Burkholderia. The use of SacB as a counter-selection method has been of limited success due to the presence of endogenous sacBC genes in the genomes of B. pseudomallei and B. mallei. These impediments have greatly hampered the genetic manipulation of B. pseudomallei and B. mallei and currently few reliable tools for the genetic manipulation of Burkholderia exist. To expand the repertoire of genetic tools for use in Burkholderia, we developed the suicide plasmid pMo130, which allows for the compliant genetic manipulation of the select agents B. pseudomallei and B. mallei using allelic exchange. pMo130 harbors an aphA gene which allows for Km selection, the reporter gene xylE, which allows for reliable visual detection of Burkholderia transformants, and carries a modified sacB gene that allows for the resolution of co-integrants. We employed this system to generate multiple unmarked and in-frame mutants in B. pseudomallei, and one mutant in B. mallei. This vector significantly expands the number of available tools that are select-agent compliant for the genetic manipulation of B. pseudomallei and B. mallei.

Role of the Pseudomonas aeruginosa PlcH Tat signal peptide in protein secretion, transcription, and cross-species Tat secretion system compatibility.

The secretion of PlcH and its homolog PlcN of Pseudomonas aeruginosa through the inner membrane depends upon a functional twin arginine translocase (Tat) system and a Tat signal sequence. Conserved twin arginine (Arg) residues within the Tat signal sequence consensus motif (S/TRRxFLK) are considered essential for the secretion of Tat substrates, but some exceptions (e.g., Lys and Arg) to the twin Arg residues in this motif have been noted. The roles of all three Arg residues within the PlcH RRRTFLK consensus motif were examined. Data are presented which indicate that Arg-9 and Arg-10 are essential for PlcH secretion across the inner membrane, but the mutation of Arg-8 (e.g., to Ala or Ser) had no observable effect on the localization of PlcH. In the signal sequence of PlcH and in all of its homologs in other bacteria, there are basic amino acid residues (Arg, Lys, and Gln) immediately adjacent to the signal peptidase cleavage site (Ala-X-Ala) that are not seen in Sec-dependent signal sequences. The mutation of these basic residues to Ala caused slightly decreased levels of extracellular PlcH, but normal localization was still observed. Deletion of the entire Tat signal sequence of PlcH not only resulted in the absence of detectable extracellular PlcH activity and protein but also caused a substantial decrease in the detectable level of plcH mRNA. Finally, data are presented which indicate that P. aeruginosa PlcH exhibits cross-species compatibility with the Escherichia coli Tat secretion machinery, but only when the E. coli Tat machinery is expressed in a P. aeruginosa host.