Overexpressed recombinant quorum quenching lactonase reduces the virulence,motility and biofilm formation of multidrug-resistant Pseudomonas aeruginosa clinical isolates

The increasing occurrence of resistance among Pseudomonas aeruginosa clinical isolates necessitates finding alternatives to antibiotics for controlling the infection of such pathogenic bacteria. In this study, lactonase gene ahl-1 from Bacillus weihenstephanensis isolate-P65 was successfully cloned and expressed in Escherichia coli BL21 (DE3) under the control of T7 promoter for utilizing its quorum quenching activity against three multidrug-resistant (MDR) P. aeruginosa clinical isolates. The biological activity of the overexpressed lactonase enzyme (Ahl-1), tested using a synthetic signal and Chromobacterium violaceum CV026 as a biosensor, displayed good catalytic activity using hexanoyl homoserine lactone (HHL) as a substrate and Chromobacterium violaceum (CV026) as a biosensor (77.2 and 133 nm min⁻¹ for the crude and the purified Ahl-lactonase enzymes, respectively). Upon challenging its ability to inhibit the virulence of three MDR P. aeruginosa clinical isolates, recombinant Ahl-1 successfully prevented the accumulation of acylhomoserine lactone signals resulting in a significant reduction in the investigated virulence determinants; protease (from 40 up to 75.5%), pyocyanin (48–75.9%), and rhamnolipids (52.7–63.4%) (P value < 0.05). Ahl-1 also displayed significant inhibitory activities on the swarming motility and biofilm formation of the three tested MDR P. aeruginosa clinical isolates (P value < 0.05). Consequently, Ahl-1 lactonase enzyme in this study is considered a promising therapeutic agent to inhibit P. aeruginosa pathogenicity with no fear of emergence of resistance.

     

Effects of magnesium deficiency on photosynthesis and carbohydrate partitioning

Magnesium nutrition is often forgotten, while its absence adversely affects numerous functions in plants. Magnesium deficiency is a growing concern for crop production frequently observed in lateritic and leached acid soils. Competition with other cations (Ca²⁺, Na⁺, and K⁺) is also found to be an essential factor, inducing magnesium deficiency in plants. This nutrient is required for chlorophyll formation and plays a key role in photosynthetic activity. Moreover, it is involved in carbohydrate transport from source-to-sink organs. Hence, sugar accumulation in leaves that results from the impairment of their transport in phloem is considered as an early response to Mg deficiency. The most visible effect is often recorded in root growth, resulting in a significant reduction of root/shoot ratio. Carbohydrate accumulation in source leaves is attributed to the unique chemical proprieties of magnesium. As magnesium is a nutrient with high mobility in plants, it is preferentially transported to source leaves to prevent severe declines in photosynthetic activity. In addition, Mg is involved in the source-to-sink transport of carbohydrates. Hence, an inverse relationship between Mg shortage and sugar accumulation in leaves is often observed. We hereby review all these aspects with a special emphasis on the role of Mg in photosynthesis and the structural and functional effects of its deficiency on the photosynthetic apparatus.     

Prosthetic valve endocarditis: management strategies and prognosis

Background Prosthetic valve endocarditis (PVE) is a rare and serious complication after heart valve replacement; its optimal management strategy, though, still needs to be defined. Objective To study the clinical, microbiological and echocardiographic characteristics of PVE and to analyse the influence of the adopted therapeutic strategy (medical or surgical) on short- and midterm outcome in a tertiary care centre in a developing country (Tunisia). Methods All cases of PVE treated in our institution between 1997 and 2006 were retrospectively analysed according to the modified DUKE criteria. Results A total of 48 PVE episodes were diagnosed (30 men and 18 women), mean age was 37.93 years. Twenty-eight patients (58.33%) were exclusively medically treated, whereas 20 (41.66%) were treated by a combined surgical and medical strategy. Indications for surgery were haemodynamic deterioration in eight patients (40%), annular abscess in six (30%) and persisting sepsis in six (30%). In comparison with those from the medical group, operated patients had a longer delay to diagnosis (p=0.025), were more frequently in heart failure (p=0.04) and experienced more early complications (p=0.011); they also more frequently had prosthetic dehiscence (p=0.015), annular abscesses (p=0.039) and vegetations >10 mm (p=0.008). Conversely, no differences were found between the groups in terms of age, sex, or nature of involved organisms. In-hospital mortality for the medical group was 14.28% and for the surgical group 35% (p=0.09). Conclusion PVE is a very serious condition carrying high mortality rates regardless of the adopted strategy. Our study demonstrates that, in selected patients, medical treatment could be a successful and acceptable approach. (Neth Heart J 2009;17: 56-60.)     

Quantitative Genome-Wide Genetic Interaction Screens Reveal Global Epistatic Relationships of Protein Complexes in Escherichia coli

Large-scale proteomic analyses in Escherichia coli have documented the composition and physical relationships of multiprotein complexes, but not their functional organization into biological pathways and processes. Conversely, genetic interaction (GI) screens can provide insights into the biological role(s) of individual gene and higher order associations. Combining the information from both approaches should elucidate how complexes and pathways intersect functionally at a systems level. However, such integrative analysis has been hindered due to the lack of relevant GI data. Here we present a systematic, unbiased, and quantitative synthetic genetic array screen in E. coli describing the genetic dependencies and functional cross-talk among over 600,000 digenic mutant combinations. Combining this epistasis information with putative functional modules derived from previous proteomic data and genomic context-based methods revealed unexpected associations, including new components required for the biogenesis of iron-sulphur and ribosome integrity, and the interplay between molecular chaperones and proteases. We find that functionally-linked genes co-conserved among γ-proteobacteria are far more likely to have correlated GI profiles than genes with divergent patterns of evolution. Overall, examining bacterial GIs in the context of protein complexes provides avenues for a deeper mechanistic understanding of core microbial systems.     

The activity of ribosome modulation factor during growth of <Emphasis Type="Italic">Escherichia coli</Emphasis> under acidic conditions

Expression of the gene encoding ribosome modulation factor (RMF), as measured using an rmf-lacZ gene fusion, increased with decreasing pH in exponential phase cultures of Escherichia coli. Expression was inversely proportional to the growth rate and independent of the acidifying agent used and it was concluded that expression of rmf was growth rate controlled in exponential phase under acid conditions. Increased rmf expression during exponential phase was not accompanied by the formation of ribosome dimers as occurs during stationary phase. Nor did it appear to have a significant effect on cell survival under acid stress since the vulnerability of an RMF-deficient mutant strain was similar to that of the parent strain. Ribosome degradation was increased in the mutant strain compared to the parent strain at pH 3.75. Also, the peptide elongation rate was reduced in the mutant strain but not the parent during growth under acid conditions. It is speculated that the function of RMF during stress-induced reduction in growth rate is two-fold: firstly to prevent reduced elongation efficiency by inactivating surplus ribosomes and thus limiting competition for available protein synthesis factors, and secondly to protect inactivated ribosomes from degradation.     

The ClpP double ring tetradecameric protease exhibits plastic ring-ring interactions, and the N termini of its subunits form flexible loops that are essential for ClpXP and ClpAP complex formation

ClpP is a conserved serine-protease with two heptameric rings that enclose a large chamber containing the protease active sites. Each ClpP subunit can be divided into a handle region, which mediates ring-ring interactions, and a head domain. ClpP associates with the hexameric ATPases ClpX and ClpA, which can unfold and translocate substrate proteins through the ClpP axial pores into the protease lumen for degradation. We have determined the x-ray structure of Streptococcus pneumoniae ClpP(A153P) at 2.5 A resolution. The structure revealed two novel features of ClpP which are essential for ClpXP and ClpAP functional activities. First, the Ala --> Pro mutation disrupts the handle region, resulting in an altered ring-ring dimerization interface, which, in conjunction with biochemical data, demonstrates the unusual plasticity of this region. Second, the structure shows the existence of a flexible N-terminal loop in each ClpP subunit. The loops line the axial pores in the ClpP tetradecamer and then protrude from the protease apical surface. The sequence of the N-terminal loop is highly conserved in ClpP across all kingdoms of life. These loops are essential determinants for complex formation between ClpP and ClpX/ClpA. Mutation of several amino acid residues in this loop or the truncation of the loop impairs ClpXP and ClpAP complex formation and prevents the coupling between ClpX/ClpA and ClpP activities.     

Residual activity and population effects of noviflumuron for German cockroach (Dictyoptera: Blattellidae) control

A benzoylphenyl urea insect growth regulator with the common name noviflumuron was evaluated for efficacy and residual activity on the German cockroach, Blattella germanica (L.). In laboratory studies evaluating residual activity, 0.05% noviflumuron suspension concentrate produced 100% nymphal mortality 120 d after application to steel and masonite substrates. Residual activity of noviflumuron was more variable on painted plywood substrates compared with stainless steel and masonite. In bioassay arenas, population reductions caused by noviflumuron were significantly greater than Archer and the untreated populations. After 16 wk, populations exposed to 0.05, 0.1, and 0.2% noviflumuron were reduced by 51.9 +/- 19.8, 62.2 +/- 6.5, and 62.6 +/- 18.4%, respectively. Control cockroach populations and populations exposed to 1.3% pyriproxyfen at labeled rate (Archer, 0.61 g/m2) increased by 1286.3 +/- 125.1 and 937.2 +/- 137.1%, respectively, at the end of 16 wk. A field study in multifamily housing complexes showed noviflumuron (0.2 and 0.5%) to provide 73.3 +/- 8.0 and 90.6 +/- 3.6% trap catch reduction at 4 wk posttreatment, respectively. There were no significant differences in the performance of noviflumuron, Maxforce FC Roach Bait Stations (0.05% [AI] fipronil), and Avert dust bait (0.05% [AI] abamectin B1). Noviflumuron shows excellent potential for use in cockroach management programs.     

Survival and feeding responses of Anacanthotermes ochraceus (Hodotermitidae: Isoptera) to local and imported wood

Forty-six local and imported wood were tested for resistance to feeding damage by the termite Anacanthotermes ochraceus (Burmeister), the most dominant species in the United Arab Emirates and the Arab Gulf region. Wood was used for construction, wall paneling, and furniture. Wood was evaluated in a 4-wk forced feeding bioassay. Each wood block was graded by the amount of termite damage by using a damage rating index (DRI) of 0 to 5 and wood rating index from very resistant to very susceptible wood. Local wood was mostly susceptible to feeding of termites; imported wood varied in resistance to feeding damage. Wood was placed in groups according to the percentages of weight loss (WL), termite survival (TS), and DRI. Wood was classified as very resistant (%WL from 0.0 to 0.3, %TS from 0.01 to 0.5, and DRI of 0.01), resistant (%WL from 1.1 to 4.9, %TS from 0.8 to 4.8, and DRI of 1.0), moderately resistant (%WL from 6.6 to 9.3, %TS from 6.3 to 8.3, and DRI of 2.0-2.3), slightly resistant (%WL from 10.1 to 19.9, %TS from 9.5 to 28.0, and DRI of 2.5-3.5), susceptible (%WL from 21.5 to 48.6, %TS from 37.3 to 64.8, and DRI of 4.0-4.3) and very susceptible (%WL from 50.0 to 59.8, %TS from 72.8 to 79.0, and DRI of 4.5-5.0). The characterization of the extracts of resistant wood may prove of economic value and lead to the development of new chemicals (repellents or antifeedants) for termite control.