Functional effects of variants of the RNA chaperone Hfq

The ring-shaped RNA chaperone Hfq has recently received much attention owing to its multiple roles in RNA metabolism. In this study we have performed a mutational analysis of the Escherichia coli hfq gene, and have studied the effects of amino acid substitutions at several positions in the Hfq protein as well as of C-terminal truncations on its role in phage Qbeta replication, in repression of a target mRNA, and on the stability of the small regulatory RNA DsrA. These functional studies provided insights into the interaction of Hfq with RNA and suggested a role for the C-terminus of Hfq in DsrA stability.     

Hyper-recombinogenic RecA protein from Pseudomonas aeruginosa with enhanced activity of its primary DNA binding site

According to one prominent model, each protomer in the activated nucleoprotein filament of homologous recombinase RecA possesses two DNA-binding sites. The primary site binds (1) single-stranded DNA (ssDNA) to form presynaptic complex and (2) the newly formed double-stranded (ds) DNA whereas the secondary site binds (1) dsDNA of a partner to initiate strand exchange and (2) the displaced ssDNA following the strand exchange. RecA protein from Pseudomonas aeruginosa (RecAPa) promotes in Escherichia coli hyper-recombination in an SOS-independent manner. Earlier we revealed that RecAPa rapidly displaces E.coli SSB protein (SSB-Ec) from ssDNA to form presynaptic complex. Here we show that this property (1) is based on increased affinity of ssDNA for the RecAPa primary DNA binding site while the affinity for the secondary site remains similar to that for E.coli RecA, (2) is not specific for SSB-Ec but is also observed for SSB protein from P.aeruginosa that, in turn, predicts a possibility of enhanced recombination repair in this pathogenic bacterium.     

In vitro trials of some antimicrobial combinations against Staphylococcus aureus and Pseudomonas aeruginosa

Staphylococcus aureus and Pseudomonas aeruginosa are rapidly increasing as multidrug resistant strains worldwide. In nosocomial settings because of heavy exposure of different antimicrobials, resistance in these pathogens turned into a grave issue in both developed and developing countries. The aim of this study was to investigate in vitro antibiotic synergism of combinations of β-lactam–β-lactam and β-lactam–aminoglycoside against clinical isolates of S. aureus and P. aeruginosa. Synergy was determined by checkerboard double dilution method. The combination of amoxicillin and cefadroxil was found to be synergistic against 47 S. aureus isolates, in the FICI range of 0.14–0.50 (81.03%) followed by the combination of streptomycin and cefadroxil synergistic against 44 S. aureus isolates in the FICI range of 0.03–0.50 (75.86%). The combination of streptomycin and cefadroxil was observed to be synergistic against 39 P. aeruginosa isolates in the FICI range of 0.16–0.50 (81.28%). Further actions are needed to characterize the possible interaction mechanism between these antibiotics. Moreover, the combination of streptomycin and cefadroxil may lead to the development of a new and vital antimicrobial against simultaneous infections of S. aureus and P. aeruginosa.     

Characterization of a rhodanese from the cyanogenic bacterium Pseudomonas aeruginosa

Pseudomonas aeruginosa, the rRNA group I type species of genus Pseudomonas, is a Gram-negative, aerobic bacterium responsible for serious infection in humans. P. aeruginosa pathogenicity has been associated with the production of several virulence factors, including cyanide. Here, the biochemical characterization of recombinant P. aeruginosa rhodanese (Pa RhdA), catalyzing the sulfur transfer from thiosulfate to a thiophilic acceptor, e.g., cyanide, is reported. Sequence homology analysis of Pa RhdA predicts the sulfur-transfer reaction to occur through persulfuration of the conserved catalytic Cys230 residue. Accordingly, the titration of active Pa RhdA with cyanide indicates the presence of one extra sulfur bound to the Cys230 Sgamma atom per active enzyme molecule. Values of K(m) for thiosulfate binding to Pa RhdA are 1.0 and 7.4mM at pH 7.3 and 8.6, respectively, and 25 degrees C. However, the value of K(m) for cyanide binding to Pa RhdA (=14 mM, at 25 degrees C) and the value of V(max) (=750 micromol min(-1)mg(-1), at 25 degrees C) for the Pa RhdA-catalyzed sulfur-transfer reaction are essentially pH- and substrate-independent. Therefore, the thiosulfate-dependent Pa RhdA persulfuration is favored at pH 7.3 (i.e., the cytosolic pH of the bacterial cell) rather than pH 8.6 (i.e., the standard pH for rhodanese activity assay). Within this pH range, conformational change(s) occur at the Pa RhdA active site during the catalytic cycle. As a whole, rhodanese may participate in multiple detoxification mechanisms protecting P. aeruginosa from endogenous and environmental cyanide.     

First Report of OXA-4, an ESBL Isolated from Pseudomonas aeruginosa a South Indian Strain

The OXA-type β-lactamases are so named because of their oxacillin-hydrolyzing abilities. In this study we characterize an extended spectrum β-lactamase, designated OXA-4, produced by a clinical isolate of Pseudomonas aeruginosa. ESBL production was detected by double disk synergy test. The P. aeruginosa isolate was obtained from endotracheal suction tip of 84 years old male patient diagnosed with CVA and hypertension. ESBL producing OXA β-lactamases was detected by PCR with primers specific to the conserved regions of the coding genes. Iso electric focusing was done to confirm the significance, sequencing the amplified product was also done. In the phenotypic identification, the strain was highly resistant to third generation cephalosporins and also to imipenem. The PCR amplified product for OXA β-lactamase was viewed at 919 bp. The pI point for the same was identified at 7.2. With the help of sequencing the amplified OXA β-lactamase was identified as OXA-4 gene. Here we report P. aeruginosa producing OXA-4 ESBL for the first time in the Indian subcontinent.     

Comparative proteomic analysis of planktonic and immobilized Pseudomonas aeruginosa cells: a multivariate statistical approach

The protein maps of Pseudomonas aeruginosa cells from two natural (attached) and one artificial (gel-entrapped) immobilized-cell (IC) systems, together with their free (suspended) counterparts, were compared after incubation for 18 or 48 h in a minimal salt medium. Principal component analysis (PCA) was used to interpret the variations in protein spot densities that were observed on electropherogram obtained by two-dimensional electrophoresis (2-DE). PCA of the 2-DE data, a matrix of 933 rows (observations, i.e., spot density values) and 12 columns (variables, i.e., incubation conditions), in which observations were standardized horizontally, extracted four principal components (PCs) accounting for 78.75% of the variability in the protein expression profiles. PC1 opposed the two modes of growth (planktonic and immobilized) while PC2 discriminated between the incubation times of free cell cultures. The incubation conditions of ICs, including the immobilization procedure (entrapment vs attachment) and the nature of the biofilm substratum, were fairly separated in PC3xPC4. The dependence of the protein patterns on the cell immobilization process was further illustrated by the identification of a number of peptides whose amount remained unchanged or was altered in ICs compared to free bacteria. These results reinforce the topical assertion that bacteria in the immobilized state display a specific physiological behavior but also question the existence of a unique IC phenotype.     

Characterization of the lipopolysaccharide from a wbjE mutant of the serogroup O11 Pseudomonas aeruginosa strain, PA103

The lipopolysaccharide (LPS) of a wbjE mutant of Pseudomonas aeruginosa PA103, a serogroup O11 strain consists of both high and low molecular weight (HMW and LMW) LPSs. The HMW LPS consisted exclusively of rhamnan A-band LPS and no B-band LPS was detected in the wbjE mutant. Interestingly, the LMW LPS from the wbjE mutant showed that it contained a variety of oligosaccharides, each with two or three phosphate groups present as mono- or pyrophosphates. These oligosaccharides consisted of the complete core octasaccharide. The GalN residue was present as an N-acetylated residue in all of these oligosaccharides except the tetrasaccharide in which it is present as an N-alanylated residue. None of these oligosaccharides contained either a d- or l-FucpNAc residue. These results are discussed with regard to the role of wbjE in the biosynthesis of P. aeruginosa PA103 B-band LPS.     

Promises and pitfalls of Pseudomonas aeruginosa lipopolysaccharide as a vaccine antigen

Antibodies directed to the Pseudomonas aeruginosa lipopolysaccharide (LPS) O-antigens have clearly shown to mediate the most effective immunity to infection caused by LPS-smooth strains. Such strains are major causes of disease in immunocompromised hosts such as burn or cancer patients, individuals in intensive care units, and those who utilize extended-wear contact lenses. Yet producing an effective vaccine composed of non-toxic, immunogenic polysaccharides has been challenging. The chemical diversity among the different O-antigens representative of the 20 major serotypes, plus additional diversity among some O-antigens representing variant subtype antigens, translates into a large degree of serologic variability that increases the complexity of O-antigen specific vaccines. Further complications come from the poor immunogenicity of the major protective epitope expressed by some O-antigens, and a large degree of diversity in animal responses that preclude predicting the optimal vaccine formulation from such studies. Nonetheless human trials over the years of vaccines eliciting O-antigen immunity have been encouraging, though no vaccine has yet been fully evaluated and found to be clinically efficacious. Newer vaccine approaches such as using polysaccharide-protein conjugates and passive therapy with monoclonal or polyclonal immune sera offer some additional means to try and produce an effective immunotherapeutic reagent for this problematic pathogen.     

Isomerization and biodegradation of beta-cypermethrin by Pseudomonas aeruginosa CH7 with biosurfactant production

Pseudomonas aeruginosa CH7, isolated from activated sludge, was able not only to isomerize and degrade beta-cypermethrin but also to utilize it as the sole source of carbon and energy for growth and produce biosurfactant. The strain effectively degraded beta-cypermethrin with inocula biomass of 0.1-0.2 g L⁻¹ at 25-35 °C, pH 6-9, and a final concentration of beta-cypermethrin 25-900 mg L⁻¹. Via response surface methodology analysis, we found the optimal condition was 29.4 °C, pH 7.0, and inocula biomass of 0.15 g L⁻¹; under these conditions, about 90% of the beta-cypermethrin could be degraded within 12 days. Noticeably, biosurfactant was detected in the MSM culture of strain CH7, suggesting that the biosurfactant (rhamnolipid) could potentially enhance the degradation of beta-cypermethrin by promoting the dissolution, adsorption, and absorption of the hydrophobic compounds. Therefore, CH7 may serve as a promising strain in the bioremediation of wastewater and soil polluted by beta-cypermethrin.     

Annexin II is a novel receptor for Pseudomonas aeruginosa

Infections with Pseudomonas aeruginosa (P. aeruginosa) are critical in ventilated and poly-traumatized patients. Most important, these bacteria cause frequent and chronic pulmonary infections in patients with cystic fibrosis. Therefore, identification of molecular mechanisms that mediate the infection of mammalian cells with P. aeruginosa is urgently required. Here, we aimed to identify novel receptors that are involved in internalization of P. aeruginosa into mammalian epithelial cells. Employing SDS-PAGE purification and mass spectrometry we demonstrate that annexin II specifically binds to P. aeruginosa. The significance of the interaction of annexin II with P. aeruginosa for the infection of mammalian cells is indicated by the finding that neutralization of the ligands on P. aeruginosa by incubation of the bacteria with recombinant, soluble annexin II prevents internalization of P. aeruginosa into human epithelial cells.     

Reactive oxygen species regulate Pseudomonas aeruginosa lipopolysaccharide-induced MUC5AC mucin expression via PKC-NADPH oxidase-ROS-TGF-alpha signaling pathways in human airway epithelial cells

Mucin overproduction is a hallmark of chronic inflammatory airway diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis. Excessive production of mucin leads to airway mucus obstruction and contributes to morbidity and mortality in these diseases. The molecular mechanisms underlying mucin overproduction, however, still remain largely unknown. Here, we report that the bacterium P. aeruginosa, an important human respiratory pathogen causing cystic fibrosis, utilizes reactive oxygen species (ROS) to up-regulate MUC5AC mucin expression. Pseudomonas aeruginosa lipopolysaccharide (PA-LPS) induces production of ROS through protein kinase C (PKC)-NADPH oxidase signaling pathway in human epithelial cells. Subsequently, ROS generation by PA-LPS releases transforming growth factor-α (TGF-α), which in turn, leads to up-regulate MUC5AC expression. These findings may bring new insights into the molecular pathogenesis of P. aeruginosa infections and lead to novel therapeutic intervention for inhibiting mucin overproduction in patients with P. aeruginosa infections.     

Molecular and structural characterization of the biosurfactant produced by Pseudomonas aeruginosa DAUPE 614

Pseudomonas aeruginosa DAUPE 614 produced rhamnolipids (3.9gL(-1)) when cultivated on a medium containing glycerol and ammonium nitrate. These rhamnolipids reduced the surface tension of water to 27.3mNm(-1), with a critical micelle concentration of 13.9mgL(-1). The maximum emulsification index against toluene was 86.4%. The structure of the carbohydrate moiety of the glycolipid was determined by gas chromatography-mass spectroscopy (GC-MS) analysis allied to electrospray ionization mass spectrometry and nuclear magnetic resonance (NMR) 1D, 2D (13)C, (1)H spectroscopy. The hydroxyl fatty acids were analyzed by GC-MS as hydroxy-acetylated fatty acid methyl ester derivatives. The positions of the fatty acids in the lipid moiety were variable, with 6 mono-rhamnolipid homologues (Rha-C(10)-C(10); Rha-C(10)-C(8); Rha-C(8)-C(10); Rha-C(10)-C(12:1); Rha-C(12)-C(10); Rha-C(10)-C(12)) and 6 di-rhamnolipid homologues (Rha(2)-C(10)-C(10); Rha(2)-C(10)-C(8); Rha(2)-C(8)-C(10); Rha(2)-C(10)-C(12:1); Rha(2)-C(12)-C(10); Rha(2)-C(10)-C(12)). The ratio of Rha(2)-C(10)-C(10) to Rha-C(10)-C(10) was higher than has been reported in previous studies. Our methodology allowed us to distinguish between the isomeric pairs Rha-C(10)-C(8)/Rha-C(8)-C(10), Rha-C(10)-C(12)/Rha-C(12)-C(10), Rha(2)-C(10)-C(8)/Rha(2)-C(8)-C(10) and Rha(2)-C(12)-C(10)/Rha(2)-C(10)-C(12). For each isomeric pair, the congener with the shorter chain adjacent to the sugar was always more abundant than the congener with longer chain.     

Lack of interchangeability of Hfq-like proteins

Hfq is an RNA-binding protein that participates in the regulatory activity of small non-coding RNAs (sRNAs) in many species of bacteria. Hfq protein was first crystallized from Staphylococcus aureus and this crystal structure constitutes a hallmark for bacterial Sm-like proteins. Paradoxically, however, the functional relevance/role of S. aureus Hfq (Hfq_SA) remains uncertain, as growing evidence suggests that the hfq_SA gene is expressed at very low levels or unexpressed in many S. aureus strains. To gather further insight, in the present work we exchanged the structural portion of the hfq gene of Salmonella enterica serovar Typhimurium (hfq_STM) with hfq_SA and analyzed the effects of the replacement on various Hfq-related phenotypes. Our results show that the replacement strain – in spite of expressing Hfq_SA at levels comparable to Hfq_STM in wild-type Salmonella – behaves as an hfq null mutant in three discrete small RNA-mediated regulatory responses. These defects correlate with an abrupt reduction in the intracellular concentration of sRNAs, as observed in an hfq null mutant. Failure of Hfq_SA to protect Salmonella sRNAs from degradation suggests that Hfq_Sa does not bind to these sRNAs. A parallel study with the Borrelia burgdorferi hfq gene (hfq_BB) gave essentially identical results: when made from a single copy chromosomal gene, Hfq_BB fails to substitute for Hfq_STM in sRNA-mediated regulation.