Comprehensive genomic analysis of an indigenous Pseudomonas pseudoalcaligenes degrading phenolic compounds.

Environmental contamination with aromatic compounds is a universal challenge. Aromatic-degrading microorganisms isolated from the same or similar polluted environments seem to be more suitable for bioremediation. Moreover, microorganisms adapted to contaminated environments are able to use toxic compounds as the sole sources of carbon and energy. An indigenous strain of Pseudomonas, isolated from the Mahshahr Petrochemical plant in the Khuzestan province, southwest of Iran, was studied genetically. It was characterized as a novel Gram-negative, aerobic, halotolerant, rod-shaped bacterium designated Pseudomonas YKJ, which was resistant to chloramphenicol and ampicillin. Genome of the strain was completely sequenced using Illumina technology to identify its genetic characteristics. MLST analysis revealed that the YKJ strain belongs to the genus Pseudomonas indicating the highest sequence similarity with Pseudomonas pseudoalcaligenes strain CECT 5344 (99% identity). Core- and pan-genome analysis indicated that P. pseudoalcaligenes contains 1,671 core and 3,935 unique genes for coding DNA sequences. The metabolic and degradation pathways for aromatic pollutants were investigated using the NCBI and KEGG databases. Genomic and experimental analyses showed that the YKJ strain is able to degrade certain aromatic compounds including bisphenol A, phenol, benzoate, styrene, xylene, benzene and chlorobenzene. Moreover, antibiotic resistance and chemotaxis properties of the YKJ strain were found to be controlled by two-component regulatory systems.

Microbiota in a cooling-lubrication circuit and an option for controlling triethanolamine biodegradation.

Cooling and lubrication agents like triethanolamine (TEA) are essential for many purposes in industry. Due to biodegradation, they need continuous replacement, and byproducts of degradation may be toxic. This study investigates an industrial (1,200 m³) cooling-lubrication circuit (CLC) that has been in operation for 20 years and is supposedly in an ecological equilibrium, thus offering a unique habitat. Next-generation (Illumina Miseq 16S rRNA amplicon) sequencing was used to profile the CLC-based microbiota and relate it to TEA and bicine dynamics at the sampling sites, influent, machine rooms, biofilms and effluent. Pseudomonas pseudoalcaligenes dominated the effluent and influent sites, while Alcaligenes faecalis dominated biofilms, and both species were identified as the major TEA degrading bacteria. It was shown that a 15 min heat treatment at 50°C was able to slow down the growth of both species, a promising option to control TEA degradation at large scale.

Quantitative proteomic analysis of Pseudomonas pseudoalcaligenes CECT5344 in response to industrial cyanide-containing wastewaters using Liquid Chromatography-Mass Spectrometry/Mass Spectrometry (LC-MS/MS).

Biological treatments to degrade cyanide are a powerful technology for cyanide removal from industrial wastewaters. It has been previously demonstrated that the alkaliphilic bacterium Pseudomonas pseudoalcaligenes CECT5344 is able to use free cyanide and several metal-cyanide complexes as the sole nitrogen source. In this work, the strain CECT5344 has been used for detoxification of the different chemical forms of cyanide that are present in alkaline wastewaters from the jewelry industry. This liquid residue also contains large concentrations of metals like iron, copper and zinc, making this wastewater even more toxic. To elucidate the molecular mechanisms involved in the bioremediation process, a quantitative proteomic analysis by LC-MS/MS has been carried out in P. pseudoalcaligenes CECT5344 cells grown with the jewelry residue as sole nitrogen source. Different proteins related to cyanide and cyanate assimilation, as well as other proteins involved in transport and resistance to metals were induced by the cyanide-containing jewelry residue. GntR-like regulatory proteins were also induced by this industrial residue and mutational analysis revealed that GntR-like regulatory proteins may play a role in the regulation of cyanide assimilation in P. pseudoalcaligenes CECT5344. The strain CECT5344 has been used in a batch reactor to remove at pH 9 the different forms of cyanide present in industrial wastewaters from the jewelry industry (0.3 g/L, ca. 12 mM total cyanide, including both free cyanide and metal-cyanide complexes). This is the first report describing the biological removal at alkaline pH of such as elevated concentration of cyanide present in a heterogeneous mixture from an industrial source.

DNA microarray analysis of the cyanotroph Pseudomonas pseudoalcaligenes CECT5344 in response to nitrogen starvation, cyanide and a jewelry wastewater.

Pseudomonas pseudoalcaligenes CECT5344 is an alkaliphilic bacterium that can use cyanide as nitrogen source for growth, becoming a suitable candidate to be applied in biological treatment of cyanide-containing wastewaters. The assessment of the whole genome sequence of the strain CECT5344 has allowed the generation of DNA microarrays to analyze the response to different nitrogen sources. The mRNA of P. pseudoalcaligenes CECT5344 cells grown under nitrogen limiting conditions showed considerable changes when compared against the transcripts from cells grown with ammonium; up-regulated genes were, among others, the glnK gene encoding the nitrogen regulatory protein PII, the two-component ntrBC system involved in global nitrogen regulation, and the ammonium transporter-encoding amtB gene. The protein coding transcripts of P. pseudoalcaligenes CECT5344 cells grown with sodium cyanide or an industrial jewelry wastewater that contains high concentration of cyanide and metals like iron, copper and zinc, were also compared against the transcripts of cells grown with ammonium as nitrogen source. This analysis revealed the induction by cyanide and the cyanide-rich wastewater of four nitrilase-encoding genes, including the nitC gene that is essential for cyanide assimilation, the cyanase cynS gene involved in cyanate assimilation, the cioAB genes required for the cyanide-insensitive respiration, and the ahpC gene coding for an alkyl-hydroperoxide reductase that could be related with iron homeostasis and oxidative stress. The nitC and cynS genes were also induced in cells grown under nitrogen starvation conditions. In cells grown with the jewelry wastewater, a malate quinone:oxidoreductase mqoB gene and several genes coding for metal extrusion systems were specifically induced.

Protective role of the neuropeptide urocortin II against experimental sepsis and leishmaniasis by direct killing of pathogens.

We currently face an alarming resurgence in infectious diseases characterized by antimicrobial resistance and therapeutic failure. This has generated the urgent need of developing new therapeutic approaches that include agents with nontraditional modes of action. A recent interest focused on approaches based on our natural immune defenses, especially on peptides that combine innate antimicrobial activity against diverse pathogens and immunoregulatory functions. In this study, to our knowledge, we describe for the first time the antimicrobial activity of the neuropeptide urocortin II (UCNII) against a panel of Gram-positive and Gram-negative bacteria and tropical parasites of the genus Leishmania. Importantly, this cytotoxicity was selective for pathogens, because UCNII did not affect mammalian cell viability. Structurally, UCNII has a cationic and amphipathic design that resembles antimicrobial peptides. Using mutants and UCNII fragments, we determined the structural requirements for the interaction between the peptide and the surface of pathogen. Following its binding to pathogen, UCNII caused cell death through different membrane-disrupting mechanisms that involve aggregation and membrane depolarization in bacteria and pore formation in Leishmania. Noteworthily, UCNII killed the infective form of Leishmania major even inside the infected macrophages. Consequently, UCNII prevented mortality caused by polymicrobial sepsis and ameliorated pathological signs of cutaneous leishmaniasis. Besides its presence in body physical and mucosal barriers, we found that innate immune cells produce UCNII in response to infections. Therefore, UCNII could be considered as an ancient highly-conserved host peptide involved in the natural antimicrobial defense and emerge as an attractive alternative to current treatments for microbial disorders with associated drug resistances.

Effect of aluminium and copper on biofilm development of Pseudomonas pseudoalcaligenes KF707 and P. fluorescens as a function of different media compositions.

Bioremediation efforts worldwide are faced with the problem of metals interfering with the degradation of organic pollutants. There has been little systematic investigation into how the important environmental factors of media composition, buffering agent, and carbon source affect the exertion of metal toxicity on bacteria. This study aimed to systematically separate and investigate the influence of these factors by examining planktonic and biofilm establishment and growth. Two Pseudomonads were chosen, the PCB degrader P. pseudoalcaligenes KF707 and P. fluorescens. The two strains were grown in the presence of Al(3+) and Cu(2+) under different media conditions of carbon source (Lysogeny broth, biphenyl, succinate, aspartic acid, butyric acid, oxaloacetic acid, putrescine and benzoic acid) and under different buffering conditions (high and low phosphate or MOPS). These experiments allowed for the elucidation of an effect of different metabolic conditions and metal speciation on planktonic bacteria growth and biofilm establishment and development under metal stress. Here we show that the nature of bacterial growth (planktonic and biofilm development) is dramatically affected by the interplay between toxic metals, carbon source and media composition. The capacity of a media to bind toxic metals as well as quality of carbon source greatly influences the amount of metal that bacteria can tolerate, depending on both the bacterium and metal. Future studies evaluating metal ion toxicity should consider these effects, as well as their interactions with specific environments into account in order to improve clean-up success.

Metabolic adaptation of Pseudomonas pseudoalcaligenes CECT5344 to cyanide: role of malate-quinone oxidoreductases, aconitase and fumarase isoenzymes.

In general, the biodegradation of a toxic compound by a micro-organism requires the concurrence of, at least, two features in the biological system: first, the capability of the micro-organism to metabolize the toxic compound, and secondly, the capacity to resist its toxic effect. Pseudomonas pseudoalcaligenes CECT5344 is a bacterium used in the biodegradation of cyanide because it is capable to use it as a nitrogen source. The present review is mainly focused on the putative role of iron-containing enzymes of the tricarboxylic acid cycle in cyanide resistance by P. pseudoalcaligenes CECT5344.

Role of Fur on cyanide tolerance of Pseudomonas pseudoalcaligenes CECT5344.

Pseudomonas pseudoalcaligenes CECT5344 can be used in cyanide bioremediation processes because it grows at pH 9.5 using 2.0 mM cyanide at the sole nitrogen source. Cyanide strongly binds to metals creating iron-deprivation conditions. The bacterium responds to the presence of cyanide by inducing several processes such as siderophore synthesis for iron capture, cyanide-insensitive respiration system and defence mechanisms against oxidative stress. Since high concentrations of cyanide cause iron deficiency and because iron is an essential nutrient, bacterial growth in the presence of cyanide requires an efficient iron uptake. Fur is a global transcription factor that regulates a diversity of biological processes such as iron homoeostasis, TCA (tricarboxylic acid) cycle metabolism and oxidative stress response. Fur's regulation of iron uptake and storage genes should play a significant role in the lives of these bacteria. In the present review, current knowledge of Fur is summarized.

Metabolomic investigation of the bacterial response to a metal challenge.

Pseudomonas pseudoalcaligenes KF707 is naturally resistant to the toxic metalloid tellurite, but the mechanisms of resistance are not known. In this study we report the isolation of a KF707 mutant (T5) with hyperresistance to tellurite. In order to characterize the bacterial response and the pathways leading to tolerance, we utilized Phenotype MicroArray technology (Biolog) and a metabolomic technique based on nuclear magnetic resonance spectroscopy. The physiological states of KF707 wild-type and T5 cells exposed to tellurite were also compared in terms of viability and reduced thiol content. Our analyses showed an extensive change in metabolism upon the addition of tellurite to KF707 cultures as well as different responses when the wild-type and T5 strains were compared. Even in the absence of tellurite, T5 cells displayed a "poised" physiological status, primed for tellurite exposure and characterized by altered intracellular levels of glutathione, branched-chain amino acids, and betaine, along with increased resistance to other toxic metals and metabolic inhibitors. We conclude that hyperresistance to tellurite in P. pseudoalcaligenes KF707 is correlated with the induction of the oxidative stress response, resistance to membrane perturbation, and reconfiguration of cellular metabolism.

Pseudomonas pseudoalcaligenes KF707 upon biofilm formation on a polystyrene surface acquire a strong antibiotic resistance with minor changes in their tolerance to metal cations and metalloid oxyanions.

The susceptibility to various biocides was examined in planktonic cells and biofilms of the obligate aerobe, PCBs degrader, Pseudomonas pseudoalcaligenes KF707. The toxicity of two antibiotics, amikacin and rifampicin, three metalloid oxyanions (AsO2(-), SeO3(2-), TeO3(2-)) and three metal cations (Cd2+, Ni2+, Al3+) was tested at two stages of the biofilm-development (4 and 24 h) and compared to planktonic cells susceptibility. Mature biofilms formed in rich (LB, Luria-Bertani) medium were thicker (23 microm) than biofilms grown in minimal (SA saccarose-arginine) medium (13 microm). Early grown (4 h) SA-biofilms, which consisted of a few sparse/attached cells, were 50-100 times more resistant to antibiotics than planktonic cells. Conversely, minor changes in tolerance to metal(loid)s were seen in both SA- and LB-grown biofilms. In contrast to planktonic cells, no reduction of TeO3(2-) to elemental Te0 or SeO3(2-) to elemental Se0 was seen in KF707 biofilms. The data indicate that: (a) metal tolerance in KF707 biofilms, under the growth and exposure conditions described here, is different than antibiotic tolerance; (b) KF707 planktonic cells and biofilms, are almost equally susceptible to killing by metal cations and oxyanions, and (c) biofilm-tolerance to TeO3(2-) and SeO3(2-) is not linked to metalloid reduction; this means that KF707 planktonic cells and biofilms differ in their physiology and strategy to counteract metalloid toxicity.

Essential role of cytochrome bd-related oxidase in cyanide resistance of Pseudomonas pseudoalcaligenes CECT5344.

Pseudomonas pseudoalcaligenes CECT5344 grows in minimal medium containing cyanide as the sole nitrogen source. Under these conditions, an O2-dependent respiration highly resistant to cyanide was detected in cell extracts. The structural genes for the cyanide-resistant terminal oxidase, cioA and cioB, are clustered and encode the integral membrane proteins that correspond to subunits I and II of classical cytochrome bd, although the presence of heme d in the membrane could not be detected by difference spectra. The cio operon from P. pseudoalcaligenes presents a singular organization, starting upstream of cioAB by the coding sequence of a putative ferredoxin-dependent sulfite or nitrite reductase and spanning downstream two additional open reading frames that encode uncharacterized gene products. PCR amplifications of RNA (reverse transcription-PCR) indicated the cyanide-dependent up-regulation and cotranscription along the operon. The targeted disruption of cioA eliminates both the expression of the cyanide-stimulated respiratory activity and the growth with cyanide as the nitrogen source, which suggests a critical role of this cytochrome bd-related oxidase in the metabolism of cyanide by P. pseudoalcaligenes CECT5344.

Evidence for a tellurite-dependent generation of reactive oxygen species and absence of a tellurite-mediated adaptive response to oxidative stress in cells of Pseudomonas pseudoalcaligenes KF707.

Tellurite (TeO3(2-)) is the most toxic and soluble oxyanion among tellurium (Te) compounds. The effects of the metalloid anion on the oxidative stress response of the obligate aerobe Pseudomonas pseudoalcaligenes KF707 were investigated. Cells treated with sub-lethal concentrations of TeO3(2-) showed neither adaptation to it nor cross-protection against oxidants such as 1,1'-4,4'-bipyridinium dichloride (paraquat, PQ2+), diazenedicarboxylic acid bis-N,N-dimethylamide (diamide), tert-butyl hydroperoxide (tBH) and hydrogen peroxide (H2O2). Notably, TeO3(2-) exerted a synergic effect on the toxicity of these latter oxidants. Tellurite was shown to decrease the cellular content of reduced thiols (RSH) with a consequent increase in the production of reactive oxygen species (ROS) and stimulation of the superoxide dismutase (SOD) activity. However, since the time course of ROS production by TeO3(2) (t1/2 > 30 min) was much slower than that with PQ2+ and/or diamide (t1/2