Identification of QuiP, the Product of Gene PA1032, as the Second Acyl-Homoserine Lactone Acylase of Pseudomonas aeruginosa PAO1

The relevance of the acyl homoserine lactone (acyl-HSL) quorum signals N-3-oxododecanoyl-homoserine lactone (3OC12HSL) and N-butanoyl-homoserine lactone to the biology and virulence of Pseudomonas aeruginosa is well investigated. Previously, P. aeruginosa was shown to degrade long-chain, but not short-chain, acyl-HSLs as sole carbon and energy sources (J. J. Huang, J.-I. Han, L.-H. Zhang, and J. R. Leadbetter, Appl. Environ. Microbiol. 69:5941-5949, 2003). A gene encoding an enzyme with acyl-HSL acylase activity, pvdQ (PA2385), was identified, but it was not required for acyl-HSL utilization. This indicated that P. aeruginosa encodes another acyl-HSL acylase, which we identify here. A comparison of total cell proteins of cultures grown with long-acyl acyl-HSLs versus other substrates implicated the involvement of a homolog of PvdQ, the product of gene PA1032, for which we propose the name QuiP. Transposon mutants of quiP were defective for growth when P. aeruginosa was cultured in medium containing decanoyl-HSL as a sole carbon and energy source. Complementation with a functional copy of quiP rescued this growth defect. When P. aeruginosa was grown in buffered lysogeny broth, constitutive expression of QuiP in P. aeruginosa led to decreased accumulations of the quorum signal 3OC12HSL, relative to the wild type. Heterologous expression of QuiP was sufficient to confer long-chain acyl-HSL acylase activity upon Escherichia coli. Examination of gene expression patterns during acyl-HSL-dependent growth of P. aeruginosa further supported the involvement of quiP in signal decay and revealed other genes also possibly involved. It is not yet known under which “natural” conditions quiP is expressed or how P. aeruginosa balances the expression of its quorum-sensing systems with the expression of its acyl-HSL acylase activities.     

The acute effects of a caffeine-containing supplement on strength, muscular endurance, and anaerobic capabilities

The purpose of this study was to examine the acute effects of a caffeine-containing supplement on upper- and lower-body strength and muscular endurance as well as anaerobic capabilities. Thirty-seven resistance-trained men (mean +/- SD, age: 21 +/- 2 years) volunteered to participate in this study. On the first laboratory visit, the subjects performed 2 Wingate Anaerobic Tests (WAnTs) to determine peak power (PP) and mean power (MP), as well as tests for 1 repetition maximum (1RM), dynamic constant external resistance strength, and muscular endurance (TOTV; total volume of weight lifted during an endurance test with 80% of the 1RM) on the bilateral leg extension (LE) and free-weight bench press (BP) exercises. Following a minimum of 48 hours of rest, the subjects returned to the laboratory for the second testing session and were randomly assigned to 1 of 2 groups: a supplement group (SUPP; n = 17), which ingested a caffeine-containing supplement, or a placebo group (PLAC; n = 20), which ingested a cellulose placebo. One hour after ingesting either the caffeine-containing supplement or the placebo, the subjects performed 2 WAnTs and were tested for 1RM strength and muscular endurance on the LE and BP exercises. The results indicated that there was a significant (p < 0.05) increase in BP 1RM for the SUPP group, but not for the PLAC group. The caffeine-containing supplement had no effect, however, on LE 1RM, LE TOTV, BP TOTV, PP, and MP. Thus, the caffeine-containing supplement may be an effective supplement for increasing upper-body strength and, therefore, could be useful for competitive and recreational athletes who perform resistance training.     

Synthesis and biological activity of N-Acylated ornithine analogues of daptomycin

N-Acylated ornithine analogues of daptomycin were synthesized and tested for their antibacterial efficacy.     

A rapid analytical method for monitoring the enantiomeric purity of chiral molecules synthesized in ionic liquid solvents

Ionic liquids (ILs) have been widely used as reaction solvents in asymmetric synthesis due to their interesting physical and chemical properties. However, monitoring reactant-to-product conversion and the enantiopurity of formed stereoisomers often involves a tedious extraction step before chromatographic analysis. In this study, a rapid and sensitive sampling method using headspace solid-phase microextraction (SPME) coupled to chiral gas chromatography was developed for the "on-line" analysis of chiral molecules in the IL solvent. Three different SPME sorbent coatings, namely polydimethylsiloxane, polyacrylate, and a polymeric ionic liquid-based fiber, were examined in this study. The analytical performance of the developed method was evaluated in terms of reproducibility, slope of calibration curve, linear range, calibration linearity, and the determination of detection limits. The SPME method was successfully applied in the determination of enantiomeric excess from selected mixtures of chiral molecules. A preliminary study was performed using an "on-fiber" derivatization approach revealing that the stereoisomers extracted by the SPME fiber can be efficiently derivatized using a short "on-fiber" derivatization step. The developed SPME method eliminates the need of sequestering the reaction, separating the compounds of interest from the IL solvent, and the addition of a derivatizing reagent.     

Reconciling extremely strong barriers with high levels of gene exchange in annual sunflowers

In several cases, estimates of gene flow between species appear to be higher than we might predict given the strength of interspecific barriers separating these species pairs. However, as far as we are aware, detailed measurements of reproductive isolation have not previously been compared with a coalescent-based assessment of gene flow. Here, we contrast these two measures in two species of sunflower, Helianthus annuus and H. petiolaris. We quantified the total reproductive barrier strength between these species by compounding the contributions of the following prezygotic and postzygotic barriers: ecogeographic isolation, reproductive asynchrony, niche differentiation, pollen competition, hybrid seed formation, hybrid seed germination, hybrid fertility, and extrinsic postzygotic isolation. From this estimate, we calculated the probability that a reproductively successful hybrid is produced: estimates of P(hyb) range from 10(-4) to 10(-6) depending on the direction of the cross and the degree of independence among reproductive barriers. We then compared this probability with population genetic estimates of the per generation migration rate (m). We showed that the relatively high levels of gene flow estimated between these sunflower species (N(e) m= 0.34-0.76) are mainly due to their large effective population sizes (N(e) > 10(6)). The interspecific migration rate (m) is very small (<10(-7)) and an order of magnitude lower than that expected based on our reproductive barrier strength estimates. Thus, even high levels of reproductive isolation (>0.999) may produce genomic mosaics.     

Analysis of Extensive [FeFe] Hydrogenase Gene Diversity Within the Gut Microbiota of Insects Representing Five Families of Dictyoptera

We have designed and utilized degenerate primers in the phylogenetic analysis of [FeFe] hydrogenase gene diversity in the gut ecosystems of roaches and lower termites. H₂ is an important free intermediate in the breakdown of wood by termite gut microbial communities, reaching concentrations in some species exceeding those measured for any other biological system. The primers designed target with specificity the largest group of enzymatic H domain proteins previously identified in a termite gut metagenome. “Family 3” hydrogenase sequences were amplified from the guts of lower termites, Incisitermes minor, Zootermopsis nevadensis, and Reticulitermes hesperus, and two roaches, Cryptocercus punctulatus and Periplaneta americana. Subsequent analyses revealed that all termite and Cryptocercus sequences were phylogenetically distinct from non-termite-associated hydrogenases available from public databases. The abundance of unique sequence operational taxonomic units (as many as 21 from each species) underscores the previously demonstrated physiological importance of H₂ to the gut ecosystems of these wood-feeding insects. The diversity of sequences observed might be reflective of multiple niches that the enzymes have been evolved to accommodate. Sequences cloned from Cryptocercus and the lower termite samples, all of which are wood feeding insects, clustered closely with one another in phylogenetic analyses to the exclusion of alleles from P. americana, an omnivorous cockroach, also cloned during this study. We present primers targeting a family of termite gut [FeFe] hydrogenases and provide results that are consistent with a pivotal role for hydrogen in the termite gut ecosystem and point toward unique evolutionary adaptations to the gut ecosystem.     

Reduced pulmonary surfactant interaction of daptomycin analogs via tryptophan replacement with alternative amino acids

Daptomycin was shown to interact in vitro with pulmonary surfactant leading to reduction of its antibacterial activity. We report herein the preparation and anti-staphylococcal activity of a series of daptomycin analogs with reduced pulmonary surfactant interaction by replacing tryptophan with various amino acids.