Blocking Phosphatidylcholine Utilization in Pseudomonas aeruginosa,via Mutagenesis of Fatty Acid,Glycerol and Choline Degradation Pathways,Confirms the Importance of This Nutrient Source In Vivo

Pseudomonas aeruginosa can grow to very high-cell-density (HCD) during infection of the cystic fibrosis (CF) lung. Phosphatidylcholine (PC), the major component of lung surfactant, has been hypothesized to support HCD growth of P. aeruginosa in vivo. The phosphorylcholine headgroup, a glycerol molecule, and two long-chain fatty acids (FAs) are released by enzymatic cleavage of PC by bacterial phospholipase C and lipases. Three different bacterial pathways, the choline, glycerol, and fatty acid degradation pathways, are then involved in the degradation of these PC components. Here, we identified five potential FA degradation (Fad) related fadBA-operons (fadBA1-5, each encoding 3-hydroxyacyl-CoA dehydrogenase and acyl-CoA thiolase). Through mutagenesis and growth analyses, we showed that three (fadBA145) of the five fadBA-operons are dominant in medium-chain and long-chain Fad. The triple fadBA145 mutant also showed reduced ability to degrade PC in vitro. We have previously shown that by partially blocking Fad, via mutagenesis of fadBA5 and fadDs, we could significantly reduce the ability of P. aeruginosa to replicate on FA and PC in vitro, as well as in the mouse lung. However, no studies have assessed the ability of mutants, defective in choline and/or glycerol degradation in conjunction with Fad, to grow on PC or in vivo. Hence, we constructed additional mutants (ΔfadBA145ΔglpD, ΔfadBA145ΔbetAB, and ΔfadBA145ΔbetABΔglpD) significantly defective in the ability to degrade FA, choline, and glycerol and, therefore, PC. The analysis of these mutants in the BALB/c mouse lung infection model showed significant inability to utilize PC in vitro, resulted in decreased replication fitness and competitiveness in vivo compared to the complement strain, although there was little to no variation in typical virulence factor production (e.g., hemolysin, lipase, and protease levels). This further supports the hypothesis that lung surfactant PC serves as an important nutrient for P. aeruginosa during CF lung infection.     

Cardiac expression and atrial fibrillation-associated remodeling of K2P2.1 (TREK-1) K channels in a porcine model

Aims

Effective management of atrial fibrillation (AF) often remains an unmet need. Cardiac two-pore-domain K⁺ (K_2P) channels are implicated in action potential regulation, and their inhibition has been proposed as a novel antiarrhythmic strategy. K_2P2.1 (TREK-1) channels are expressed in the human heart. This study was designed to identify and functionally express porcine K_2P2.1 channels. In addition, we sought to analyze cardiac expression and AF-associated K_2P2.1 remodeling in a clinically relevant porcine AF model.

Main methods

Three pK_2P2.1 isoforms were identified and amplified. Currents were recorded using voltage clamp electrophysiology in the Xenopus oocyte expression system. K_2P2.1 remodeling was studied by quantitative real time PCR and Western blot in domestic pigs during AF induced by atrial burst pacing.

Key findings

Human and porcine K_2P2.1 proteins share 99% identity. Residues involved in phosphorylation or glycosylation are conserved. Porcine K_2P2.1 channels carried outwardly rectifying K⁺ currents similar to their human counterparts. In pigs, K_2P2.1 was expressed ubiquitously in the heart with predominance in the atrial tissue. AF was associated with time-dependent reduction of K_2P2.1 protein in the RA by 70% (7 days of AF) and 80% (21 days of AF) compared to control animals in sinus rhythm. K_2P2.1 expression in the left atrium, AV node, and ventricles was not affected by AF.

Significance

Similarities between porcine and human K_2P2.1 channels indicate that the pig may represent a valid model for mechanistic and preclinical studies. AF-related atrial K_2P2.1 remodeling has potential implications for arrhythmia maintenance and antiarrhythmic therapy.     

mini-Tn7 insertion in bacteria with multiple glmS-linked attTn7 sites: example Burkholderia mallei ATCC 23344

The mini-Tn7 vectors are universally applicable in gram-negative bacteria and thereby facilitate the manipulation of many organisms for which few genetic systems are available. These vectors, when provided with only the Tn7 site-specific transposition machinery, insert site and orientation specifically in the bacterial chromosome at an attTn7 site downstream of the essential glmS gene. A few bacteria, including Burkholderia spp., contain multiple glmS genes and therefore several attTn7 sites. Here we provide a protocol for application of the mini-Tn7 system in B. mallei as an example of bacteria with multiple glmS sites. The procedure involves, first, cloning of the genes of interest into an appropriate mini-Tn7 vector; second, co-transfer of the recombinant mini-Tn7 vector and a helper plasmid encoding the Tn7 site-specific transposition pathway into B. mallei by conjugation, followed by selection of insertion-containing strains; and last, PCR verification of mini-Tn7 insertions. B. mallei possesses two glmS genes on chromosome 1 and Tn7 transposes to both sites, although transposition to attTn7-1 associated with glmS1 occurs in more than 90% of the clones examined. Transposition is efficient and the whole procedure from start to verification of insertion events can be done in less than 5 d. This first chromosome integration system in B. mallei provides an important contribution to the genetic tools emerging for Burkholderia spp. Vectors are available for gene complementation and expression, and gene fusion analyses.     

mini-Tn7 insertion in bacteria with secondary, non-glmS-linked attTn7 sites: example Proteus mirabilis HI4320

We previously constructed a series of mini-Tn7 chromosome integration vectors that, when provided only with the site-specific transposition machinery, generally transpose to a naturally evolved, neutral attTn7 site that is located 25-bp downstream of the glmS gene. Here we provide a protocol for application of the mini-Tn7 system in Proteus mirabilis as an example of a bacterium with a secondary attTn7 site that is not linked to glmS but, in this case, located in the carAB operon. The procedure involves, first, cloning of the genes of interest into an appropriate mini-Tn7 vector; second, co-transfer of the recombinant mini-Tn7 vector and a helper plasmid encoding the Tn7 site-specific transposition pathway into P. mirabilis by transformation, followed by selection of insertion-containing strains; third, PCR verification of mini-Tn7 insertions; and last, optional Flp-mediated excision of the antibiotic-resistance selection marker present on the chromosomally integrated mini-Tn7 element. When transposon-containing cells are selected on rich medium, insertions occur at both attTn7 sites with equal efficiency and frequency. Because carA mutants are arginine and pyrimidine auxotrophs, single-site insertions at the glmS attTn7 sites can be obtained by selection on minimal medium. From start to verification of the insertion events, the whole procedure takes 5 d. This chromosome integration system in P. mirabilis provides an important tool for animal and biofilm studies based on this bacterium. Vectors are available for gene complementation and expression, gene fusion analyses and tagging with a green fluorescent protein (GFP)-encoding reporter gene.     

Quorum-sensing signal synthesis by the Yersinia pestis acyl-homoserine lactone synthase YspI

The acyl-homoserine lactone molecular species (AHLs) produced by the Yersinia pestis AHL synthase YspI were identified by biochemical and physical/chemical techniques. Bioassays of extracts from culture supernatants of the recombinant YspI and wild-type Yersinia pestis showed similar profiles of AHLs. Analysis by liquid chromatography-mass spectrometry revealed that the predominant AHLs were N-3-oxooctanoyl-L-homoserine lactone and N-3-oxo-hexanoyl-L-homoserine lactone.     

Yeast-based assay for the measurement of positive and negative influences on microsatellite stability

The evolutionary conservation of mismatch repair and Saccharomyces cerevisiae as a model system have been exploited for monitoring the influence of everyday beverages and the antineoplastic agent, hydroxyurea, on the stability of regions of highly repetitive DNA known as microsatellites. Two different reporter systems are compared for sensitivity and reproducibility by measuring the extent of frame slippage events occurring in microsatellite regions in wild-type and mismatch repair-compromised yeast strains. Increased frame slippage results in increased reporter gene expression and hence represents instability within the repetitive region, whereas a decrease or no significant change indicates the faithful replication of the original assay plasmid, suggesting a beneficial or neutral effect of the test component. A significant outcome of this study was the identification of the protective influence exerted by the green tea catechin (-)-epigallocatechin-3-gallate (EGCG) against microsatellite instability, which is in agreement with the hypothesis that EGCG is the major chemopreventive ingredient of green tea. Immunological detection can also be used in conjunction with the green fluorescent protein (GFP) version of the assay system to identify compounds, such as hydroxyurea, which increased microsatellite instability. The system has the potential for development as a high-throughput assay for wider application.     

Distribution of insect pathogenic soil fungi in Switzerland with special reference to Beauveria brongniartii and Metharhizium anisopliae

Between 1998 and 2000 soil samples from 82fields in north, east, central and south westof Switzerland were analysed for presence ofinsect pathogenic soil fungi using a selectivemedium and the Galleria bait method. Thedetection rates with either method were verysimilar. 96% of the fields distributed overall examined regions contained Metarhiziumanisopliae. The presence of Beauveriabrongniartii was limited to sites colonised byits host, Melolontha melolontha. It wasalso present at a site where M.melolontha disappeared about 40 years ago. Onthe other hand, B. brongniartii was notfound in four fields containing M.melolontha populations. Beauveriabassiana, Paecilomyces fumosoroseus and Conidiobolus sp. were other entomopathogenicfungi isolated during this survey. Differencesin the presence of M. anisopliae betweenarable fields and adjacent meadows as well asbetween orchards and meadows are discussed.     

Morphology Dependence on Surface-Enhanced Raman Scattering Using Gold Nanorod Arrays Consisting of Agglomerated Nanoparticles

Plasmonics - Highly ordered arrays of vertically aligned Au nanorod arrays consisting of agglomerated nanoparticles are fabricated by porous anodic aluminum oxide (AAO) template-assisted...     

Escherichia-Pseudomonas shuttle vectors derived from pUC18/19

Two new broad-host-range plasmid vectors, pUCP18 and pUCP19, which are stably maintained in Escherichia coli and Pseudomonas aeruginosa have been constructed. The plasmids are based on the E. coli pUC18 and pUC19 vectors and possess all their features: (i) convenient direct screening of recombinants; (ii) versatile multiple cloning site; (iii) use as sequencing and expression vectors; (iv) small size; and (v) intermediate to high copy number.