Genomic Insights into Cyanide Biodegradation in the Pseudomonas Genus.

Molecular studies about cyanide biodegradation have been mainly focused on the hydrolytic pathways catalyzed by the cyanide dihydratase CynD or the nitrilase NitC. In some Pseudomonas strains, the assimilation of cyanide has been linked to NitC, such as the cyanotrophic model strain Pseudomonas pseudoalcaligenes CECT 5344, which has been recently reclassified as Pseudomonas oleovorans CECT 5344. In this work, a phylogenomic approach established a more precise taxonomic position of the strain CECT 5344 within the species P. oleovorans. Furthermore, a pan-genomic analysis of P. oleovorans and other species with cyanotrophic strains, such as P. fluorescens and P. monteilii, allowed for the comparison and identification of the cioAB and mqoAB genes involved in cyanide resistance, and the nitC and cynS genes required for the assimilation of cyanide or cyanate, respectively. While cyanide resistance genes presented a high frequency among the analyzed genomes, genes responsible for cyanide or cyanate assimilation were identified in a considerably lower proportion. According to the results obtained in this work, an in silico approach based on a comparative genomic approach can be considered as an agile strategy for the bioprospection of putative cyanotrophic bacteria and for the identification of new genes putatively involved in cyanide biodegradation.

Quantitative Proteomic Analysis of Cyanide and Mercury Detoxification by Pseudomonas pseudoalcaligenes CECT 5344.

The cyanide-degrading bacterium Pseudomonas pseudoalcaligenes CECT 5344 uses cyanide and different metal-cyanide complexes as the sole nitrogen source. Under cyanotrophic conditions, this strain was able to grow with up to 100 µM mercury, which was accumulated intracellularly. A quantitative proteomic analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been applied to unravel the molecular basis of the detoxification of both cyanide and mercury by the strain CECT 5344, highlighting the relevance of the cyanide-insensitive alternative oxidase CioAB and the nitrilase NitC in the tolerance and assimilation of cyanide, independently of the presence or absence of mercury. Proteins overrepresented in the presence of cyanide and mercury included mercury transporters, mercuric reductase MerA, transcriptional regulator MerD, arsenate reductase and arsenical resistance proteins, thioredoxin reductase, glutathione S-transferase, proteins related to aliphatic sulfonates metabolism and sulfate transport, hemin import transporter, and phosphate starvation induced protein PhoH, among others. A transcriptional study revealed that from the six putative merR genes present in the genome of the strain CECT 5344 that could be involved in the regulation of mercury resistance/detoxification, only the merR2 gene was significantly induced by mercury under cyanotrophic conditions. A bioinformatic analysis allowed the identification of putative MerR2 binding sites in the promoter regions of the regulatory genes merR5, merR6, arsR, and phoR, and also upstream from the structural genes encoding glutathione S-transferase (fosA and yghU), dithiol oxidoreductase (dsbA), metal resistance chaperone (cpxP), and amino acid/peptide extruder involved in quorum sensing (virD), among others. IMPORTANCE Cyanide, mercury, and arsenic are considered very toxic chemicals that are present in nature as cocontaminants in the liquid residues generated by different industrial activities like mining. Considering the huge amounts of toxic cyanide- and mercury-containing wastes generated at a large scale and the high biotechnological potential of P. pseudoalcaligenes CECT 5344 in the detoxification of cyanide present in these industrial wastes, in this work, proteomic, transcriptional, and bioinformatic approaches were used to characterize the molecular response of this bacterium to cyanide and mercury, highlighting the mechanisms involved in the simultaneous detoxification of both compounds. The results generated could be applied for developing bioremediation strategies to detoxify wastes cocontaminated with cyanide, mercury, and arsenic, such as those generated at a large scale in the mining industry.

Proteomic Analysis of Arsenic Resistance during Cyanide Assimilation by Pseudomonas pseudoalcaligenes CECT 5344.

Wastewater from mining and other industries usually contains arsenic and cyanide, two highly toxic pollutants, thereby creating the need to develop bioremediation strategies. Here, molecular mechanisms triggered by the simultaneous presence of cyanide and arsenite were analyzed by quantitative proteomics, complemented with qRT-PCR analysis and determination of analytes in the cyanide-assimilating bacterium Pseudomonas pseudoalcaligenes CECT 5344. Several proteins encoded by two ars gene clusters and other Ars-related proteins were up-regulated by arsenite, even during cyanide assimilation. Although some proteins encoded by the cio gene cluster responsible for cyanide-insensitive respiration decreased in the presence of arsenite, the nitrilase NitC required for cyanide assimilation was unaffected, thus allowing bacterial growth with cyanide and arsenic. Two complementary As-resistance mechanisms were developed in this bacterium, the extrusion of As(III) and its extracellular sequestration in biofilm, whose synthesis increased in the presence of arsenite, and the formation of organoarsenicals such as arseno-phosphoglycerate and methyl-As. Tetrahydrofolate metabolism was also stimulated by arsenite. In addition, the ArsH2 protein increased in the presence of arsenite or cyanide, suggesting its role in the protection from oxidative stress caused by both toxics. These results could be useful for the development of bioremediation strategies for industrial wastes co-contaminated with cyanide and arsenic.

Biodegradation of polybutylene adipate-co-terephthalate by Priestia megaterium, Pseudomonas mendocina, and Pseudomonas pseudoalcaligenes following incubation in the soil.

Soil that contained polybutylene adipate-co-terephthalate (PBAT) was incubated with Priestia megaterium, Pseudomonas mendocina, and Pseudomonas pseudoalcaligenes to improve the biodegradative process of this polymer. The mixture of Pr. megaterium and Ps. mendocina was highly effective at biodegrading the PBAT, and after eight weeks of soil incubation, approximately 84% of the PBAT film weight was lost. Mixtures of the other two species also positively affected the synergistic degradation of PBAT film in the soil, but the mixture of three species had a negative effect. The residual PBAT film microstructure clearly demonstrated the degradation of PBAT, and the degree of degradation was related to the different species. Cleavage of the PBAT film ester bond after soil microbial action affected its properties. The incubation of PBAT in soil that contained these species affected soil dehydrogenase and soil lipase in particular. The secretion of lipase by these species could play an important role in the degradation of PBAT in the soil.

Alternative Pathway for 3-Cyanoalanine Assimilation in Pseudomonas pseudoalcaligenes CECT5344 under Noncyanotrophic Conditions.

3-Cyanoalanine and cyanohydrins are intermediate nitriles produced in cyanide degradation pathways in plants and bacteria. 3-Cyanoalanine is generated from cyanide by the 3-cyanoalanine synthase, an enzyme mainly characterized in cyanogenic plants. NIT4-type nitrilases use 3-cyanoalanine as a substrate, forming ammonium and aspartate. In some organisms, this enzyme also generates asparagine through an additional nitrile hydratase activity. The alkaliphilic bacterium Pseudomonas pseudoalcaligenes CECT5344 assimilates cyanide through an intermediate cyanohydrin, which is further converted into ammonium by the nitrilase NitC. This bacterium also contains three additional nitrilases, including Nit4. In this work, a proteomic analysis of P. pseudoalcaligenes CECT5344 cells grown with 3-cyanoalanine as the sole nitrogen source has revealed the overproduction of different proteins involved in nitrogen metabolism, including the nitrilase NitC. In contrast, the nitrilase Nit4 was not induced by 3-cyanoalanine, and it was only overproduced in cells grown with a cyanide-containing jewelry-manufacturing residue. Phenotypes of single and double mutant strains defective in nit4 or/and nitC revealed the implication of the nitrilase NitC in the assimilation of 3-cyanoalanine and suggest that the 3-cyanoalanine assimilation pathway in P. pseudoalcaligenes CECT5344 depends on the presence or absence of cyanide. When cyanide is present, 3-cyanoalanine is assimilated via Nit4, but in the absence of cyanide, a novel pathway for 3-cyanoalanine assimilation, in which the nitrilase NitC uses the nitrile generated after deamination of the α-amino group from 3-cyanoalanine, is proposed. IMPORTANCE Nitriles are organic cyanides with important industrial applications, but they are also found in nature. 3-Cyanoalanine is synthesized by plants and some bacteria to detoxify cyanide from endogenous or exogenous sources, but this nitrile may be also involved in other processes such as stress tolerance, nitrogen and sulfur metabolism, and signaling. The cyanide-degrading bacterium Pseudomonas pseudoalcaligenes CECT5344 grows with 3-cyanoalanine as the sole nitrogen source, but it does not use this nitrile as an intermediate in the cyanide assimilation pathway. In this work, a quantitative proteomic analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed to study, for the first time, the response to 3-cyanoalanine at the proteomic level. Proteomic data, together with phenotypes of different nitrilase-defective mutants of P. pseudoalcaligenes CECT5344, provide evidence that in the absence of cyanide, the nitrilase Nit4 is not involved in 3-cyanoalanine assimilation, and instead, the nitrilase NitC participates in a novel alternative 3-cyanoalanine assimilation pathway.

Volatile organic compounds produced by Pseudomonas pseudoalcaligenes alleviated drought stress by modulating defense system in maize (Zea mays L.).

Research on plant growth-promoting bacteria (PGPR) revealed an effective role of bacterial volatile organic compounds (VOCs) in stress alleviation. Out of 15 PGPR strains, infection with VOCs from Pseudomonas pseudoalcaligenes' resulted in maximum germination, growth promotion, and drought tolerance in maize plants. The VOCs of P. pseudoalcaligenes caused induced systemic tolerance in maize plants during 7 days of drought stress. The VOCs exposed plants displayed resistance to drought stress by reducing electrolyte leakage and malondialdehyde content and increasing the synthesis of photosynthetic pigments, proline, and phytohormones contents. Maize plants revealed enhanced resistance by showing higher activities of antioxidant defense enzymes both in shoots and roots under drought stress. Activities of antioxidant enzymes were more pronounced in shoots than roots. Gas chromatography and mass spectrophotometric (GC-MS) analysis comparing VOCs produced by the most efficient P. pseudoalcaligenes strain and inefficient strains of Pseudomonas sp. grown in culture media revealed nine compounds that they had in common. However, dimethyl disulfide, 2,3-butanediol, and 2-pentylfuran were detected only in P. pseudoalcaligenes, indicating these compounds are potential candidates for drought stress induction. Further studies are needed to unravel the molecular mechanisms of VOCs-mediated systemic drought tolerance in plants related to each identified VOC.

Carbon Source Influence on Extracellular pH Changes along Bacterial Cell-Growth.

The effect of initial pH on bacterial cell-growth and its change over time was studied under aerobic heterotrophic conditions by using three bacterial strains: Escherichia coli ATCC 25922, Pseudomonas putida KT2440, and Pseudomonas pseudoalcaligenes CECT 5344. In Luria-Bertani (LB) media, pH evolved by converging to a certain value that is specific for each bacterium. By contrast, in the buffered Minimal Medium (MM), pH was generally more stable along the growth curve. In MM with glucose as carbon source, a slight acidification of the medium was observed for all strains. In the case of E. coli, a sudden drop in pH was observed during exponential cell growth that was later recovered at initial pH 7 or 8, but was irreversible below pH 6, thus arresting further cell-growth. When using other carbon sources in MM at a fixed initial pH, pH changes depended mainly on the carbon source itself. While glucose, glycerol, or octanoate slightly decreased extracellular pH, more oxidized carbon sources, such as citrate, 2-furoate, 2-oxoglutarate, and fumarate, ended up with the alkalinization of the medium. These observations are in accordance with pH change predictions using genome-scale metabolic models for the three strains, thus revealing the metabolic reasons behind pH change. Therefore, we conclude that the composition of the medium, specifically the carbon source, determines pH change during bacterial growth to a great extent and unravel the main molecular mechanism behind this phenotype. These findings pave the way for predicting pH changes in a given bacterial culture and may anticipate the interspecies interactions and fitness of bacteria in their environment.

Evidence for Environmental-Human Microbiota Transfer at a Manufacturing Facility with Novel Work-related Respiratory Disease.

Rationale: Workers' exposure to metalworking fluid (MWF) has been associated with respiratory disease.Objectives: As part of a public health investigation of a manufacturing facility, we performed a cross-sectional study using paired environmental and human sampling to evaluate the cross-pollination of microbes between the environment and the host and possible effects on lung pathology present among workers.Methods: Workplace environmental microbiota were evaluated in air and MWF samples. Human microbiota were evaluated in lung tissue samples from workers with respiratory symptoms found to have lymphocytic bronchiolitis and alveolar ductitis with B-cell follicles and emphysema, in lung tissue samples from control subjects, and in skin, nasal, and oral samples from 302 workers from different areas of the facility. In vitro effects of MWF exposure on murine B cells were assessed.Measurements and Main Results: An increased similarity of microbial composition was found between MWF samples and lung tissue samples of case workers compared with control subjects. Among workers in different locations within the facility, those that worked in the machine shop area had skin, nasal, and oral microbiota more closely related to the microbiota present in the MWF samples. Lung samples from four index cases and skin and nasal samples from workers in the machine shop area were enriched with Pseudomonas, the dominant taxa in MWF. Exposure to used MWF stimulated murine B-cell proliferation in vitro, a hallmark cell subtype found in the pathology of index cases.Conclusions: Evaluation of a manufacturing facility with a cluster of workers with respiratory disease supports cross-pollination of microbes from MWF to humans and suggests the potential for exposure to these microbes to be a health hazard.

Halotolerant rhizobacteria Pseudomonas pseudoalcaligenes and Bacillus subtilis mediate systemic tolerance in hydroponically grown soybean (Glycine max L.) against salinity stress.

Salt stress is one of the devastating factors that hampers growth and productivity of soybean. Use of Pseudomonas pseudoalcaligenes to improve salt tolerance in soybean has not been thoroughly explored yet. Therefore, we observed the response of hydroponically grown soybean plants, inoculated with halotolerant P. pseudoalcaligenes (SRM-16) and Bacillus subtilis (SRM-3) under salt stress. In vitro testing of 44 bacterial isolates revealed that four isolates showed high salt tolerance. Among them, B. subtilis and P. pseudoalcaligenes showed ACC deaminase activity, siderophore and indole acetic acid (IAA) production and were selected for the current study. We determined that 106 cells/mL of B. subtilis and P. pseudoalcaligenes was sufficient to induce tolerance in soybean against salinity stress (100 mM NaCl) in hydroponics by enhancing plant biomass, relative water content and osmolytes. Upon exposure of salinity stress, P. pseudoalcaligenes inoculated soybean plants showed tolerance by the increased activities of defense related system such as ion transport, antioxidant enzymes, proline and MDA content in shoots and roots. The Na+ concentration in the soybean plants was increased in the salt stress; while, bacterial priming significantly reduced the Na+ concentration in the salt stressed soybean plants. However, the antagonistic results were observed for K+ concentration. Additionally, soybean primed with P. pseudoalcaligenes and exposed to 100 mM NaCl showed a new protein band of 28 kDa suggesting that P. pseudoalcaligenes effectively reduced salt stress. Our results showed that salinity tolerance was more pronounced in P. pseudoalcaligenes as compared to B. subtilis. However, a detailed study at molecular level to interpret the mechanism by which P. pseudoalcaligenes alleviates salt stress in soybean plants need to be explored.

MerP/MerT-mediated mechanism: A different approach to mercury resistance and bioaccumulation by marine bacteria.

Currently, mechanism underlying mercury resistance and bioaccumulation of marine bacteria remains little understood. A marine bacterium Pseudomonas pseudoalcaligenes S1 is resistant to 120 mg/L Hg2+ with bioaccumulation capacity of 133.33 mg/g. Accordingly, Hg2+ resistance and bioaccumulation mechanism of S1 was investigated at molecular and cellular level. Annotation of S1 transcriptome reveals 772 differentially expressed genes, including Hg2+-relevant genes merT, merP and merA. Both merT and merP gene have three complete copies in S1 genome, while merA gene has only one. In order to evaluate the function of these Hg2+-relevant genes, three recombinant strains were constructed to express MerA (named as A), MerT/MerP (TP) and MerT/MerP/MerA (TPA), respectively. The results show that Hg2+ resistance of strain TP, TPA, and A are improved with minimum inhibition concentration (MIC) being 60 mg/L, 40 mg/L, and 20 mg/L, respectively compared to 2 mg/L of host strain. Strain TP and TPA exhibit enhanced Hg2+ bioaccumulation capacity, while strain A does not differ from the control. Their equilibrium Hg2+ bioaccumulation capacities are 110.48 mg/g, 94.49 mg/g, 83.76 mg/g and 82.29 mg/g, respectively. Summarily, different from most microorganisms that exhibit Hg2+ resistance by MerA-mediated mechanism, marine bacterium S1 achieves Hg2+ resistance and bioaccumulation capability via MerT/MerP-mediated strategy.

New evolving strategies revealed by transcriptomic analysis of a fur- mutant of the cyanotrophic bacterium Pseudomonas pseudoalcaligenes CECT 5344.

The transcriptomic analysis (RNA-seq) of a fur mutant of P. pseudoalcaligenes CECT 5344 has revealed that Fur regulates the expression of more than 100 genes in this bacterial strain, most of them negatively. The highest upregulated genes in response to fur deletion, with respect to the wild type, both cultivated in LB medium, corresponded to genes implicated in iron uptake. They include both TonB-dependent siderophore transporters for the active transport across the outer membrane, and ABC-type and MSF-type transporters for the active transport across the cytoplasmic membrane. Therefore, the main response of this bacterium to iron limitation is expressing genes necessary for metabolism of Fe siderophores produced by other microorganisms (xenosiderophores). The number of genes whose expression decreased in the fur- mutant, as well as its normalized expression (fold change), was lower. Among them, it is remarkable the presence of one of the two cas operons of the two CRISP/Cas clusters was detected in the genome of this bacterium. The transcriptome was validated by qPCR, including the decrease in the expression of cas genes (cse1). The expression of cse1 was also decreased by limiting the amount of iron, carbon or nitrogen in the medium, or by adding menadione, a compound that causes oxidative stress. The higher decrease in cse1 expression was triggered by the addition of cyanide in minimal medium. These results suggest that this bacterium responds to stress conditions, and especially to cyanide, taking a reasonable risk with respect to both the uptake of (TonB-dependent receptors gates) and the tolerance to (reduced immunity) foreign nucleic acids. In conjunction, this can be considered a yet unknown molecular mechanism forcing bacterial evolution.

Phylogenetic characterization of the energy taxis receptor Aer in Pseudomonas and phenotypic characterization in Pseudomonas pseudoalcaligenes KF707.

Chemotaxis allows bacteria to sense gradients in their environment and respond by directing their swimming. Aer is a receptor that, instead of responding to a specific chemoattractant, allows bacteria to sense cellular energy levels and move towards favourable environments. In Pseudomonas, the number of apparent Aer homologues differs between the only two species it has been characterized in, Pseudomonas aeruginosa and Pseudomonas putida. Here we combined bioinformatic approaches with deletional mutagenesis in Pseudomonas pseudoalcaligenes KF707 to further characterize Aer. It was determined that the number of Aer homologues varies between zero and four throughout the genus Pseudomonas, and they were phylogenetically classified into five subgroups. We also used sequence analysis to show that these homologous receptors differ in their HAMP signal transduction domains. Genetic analysis also indicated that some Aer homologues have likely been subject to horizontal transfer. P. pseudoalcaligenes KF707 was unique among strains for having three Aer homologues as well as the receptors CttP and McpB. Phenotypic characterization in this strain showed that the most prevalent homologue of Aer was key, but not essential, for energy taxis. This study demonstrates that energy taxis in Pseudomonas varies between species and provides a new naming convention and associated phylogenetic details for Aer chemoreceptors.

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.

A Case of Adaptive Laboratory Evolution (ALE): Biodegradation of Furfural by Pseudomonas pseudoalcaligenes CECT 5344.

Pseudomonas pseudoalcaligenes CECT 5344 is a bacterium able to assimilate cyanide as a nitrogen source at alkaline pH. Genome sequencing of this strain allowed the detection of genes related to the utilization of furfurals as a carbon and energy source. Furfural and 5-(hydroxymethyl) furfural (HMF) are byproducts of sugars production during the hydrolysis of lignocellulosic biomass. Since they inhibit the yeast fermentation to obtain bioethanol from sugars, the biodegradation of these compounds has attracted certain scientific interest. P. pseudoalcaligenes was able to use furfuryl alcohol, furfural and furoic acid as carbon sources, but after a lag period of several days. Once adapted, the evolved strain (R1D) did not show any more prolonged lag phases. The transcriptomic analysis (RNA-seq) of R1D revealed a non-conservative punctual mutation (L261R) in BN5_2307, a member of the AraC family of activators, modifying the charge of the HTH region of the protein. The inactivation of the mutated gene in the evolved strain by double recombination reverted to the original phenotype. Although the bacterium did not assimilate HMF, it transformed it into value-added building blocks for the chemical industry. These results could be used to improve the production of cost-effective second-generation biofuels from agricultural wastes.

Cyanate Assimilation by the Alkaliphilic Cyanide-Degrading Bacterium Pseudomonas pseudoalcaligenes CECT5344: Mutational Analysis of the cyn Gene Cluster.

The alkaliphilic bacterium Pseudomonas pseudoalcaligenes CECT5344 can grow with cyanate, cyanide, or cyanide-containing industrial residues as the sole nitrogen source, but the assimilation of cyanide and cyanate takes place through independent pathways. Therefore, cyanide degradation involves a chemical reaction between cyanide and oxaloacetate to form a nitrile that is hydrolyzed to ammonium by the nitrilase NitC, whereas cyanate assimilation requires a cyanase that catalyzes cyanate decomposition to ammonium and carbon dioxide. The P. pseudoalcaligenes CECT5344 cynFABDS gene cluster codes for the putative transcriptional regulator CynF, the ABC-type cyanate transporter CynABD, and the cyanase CynS. In this study, transcriptional analysis revealed that the structural cynABDS genes constitute a single transcriptional unit, which was induced by cyanate and repressed by ammonium. Mutational characterization of the cyn genes indicated that CynF was essential for cynABDS gene expression and that nitrate/nitrite transporters may be involved in cyanate uptake, in addition to the CynABD transport system. Biodegradation of hazardous jewelry wastewater containing high amounts of cyanide and metals was achieved in a batch reactor operating at an alkaline pH after chemical treatment with hydrogen peroxide to oxidize cyanide to cyanate.

Putative small RNAs controlling detoxification of industrial cyanide-containing wastewaters by Pseudomonas pseudoalcaligenes CECT5344.

The alkaliphilic bacterium Pseudomonas pseudoalcaligenes CECT5344 uses free cyanide and several metal-cyanide complexes as the sole nitrogen source and tolerates high concentrations of metals like copper, zinc and iron, which are present in the jewelry wastewaters. To understand deeply the regulatory mechanisms involved in the transcriptional regulation of cyanide-containing wastewaters detoxification by P. pseudoalcaligenes CECT5344, RNA-Seq has been performed from cells cultured with a cyanide-containing jewelry wastewater, sodium cyanide or ammonium chloride as the sole nitrogen source. Small RNAs (sRNAs) that may have potential regulatory functions under cyanotrophic conditions were identified. In total 20 sRNAs were identified to be differentially expressed when compared the jewelry residue versus ammonium as nitrogen source, 16 of which could be amplified successfully by RT-PCR. As predicted targets of these 16 sRNAs were several components of the nit1C gene cluster encoding the nitrilase NitC essential for cyanide assimilation, the cioAB gene cluster that codes for the cyanide-insensitive cytochrome bd-type terminal oxidase, the medium length-polyhydroxyalkanoates (ml-PHAs) gene cluster, and gene clusters related with a global nitrogen limitation response like those coding for glutamine synthase and urease. Other targets were non-clustered genes (or their products) involved in metal resistance and iron acquisition, such as metal extrusion systems and the ferric uptake regulatory (Fur) protein, and a GntR-like regulatory family member probably involved in the regulation of the cyanide assimilation process in the strain CECT5344. Induction of genes targeted by sRNAs in the jewelry residue was demonstrated by qRT-PCR.

Response of the Biocontrol Agent Pseudomonas pseudoalcaligenes AVO110 to Rosellinia necatrix Exudate.

The rhizobacterium Pseudomonas pseudoalcaligenes AVO110, isolated by the enrichment of competitive avocado root tip colonizers, controls avocado white root rot disease caused by Rosellinia necatrix Here, we applied signature-tagged mutagenesis (STM) during the growth and survival of AVO110 in fungal exudate-containing medium with the goal of identifying the molecular mechanisms linked to the interaction of this bacterium with R. necatrix A total of 26 STM mutants outcompeted by the parental strain in fungal exudate, but not in rich medium, were selected and named growth-attenuated mutants (GAMs). Twenty-one genes were identified as being required for this bacterial-fungal interaction, including membrane transporters, transcriptional regulators, and genes related to the metabolism of hydrocarbons, amino acids, fatty acids, and aromatic compounds. The bacterial traits identified here that are involved in the colonization of fungal hyphae include proteins involved in membrane maintenance (a dynamin-like protein and ColS) or cyclic-di-GMP signaling and chemotaxis. In addition, genes encoding a DNA helicase (recB) and a regulator of alginate production (algQ) were identified as being required for efficient colonization of the avocado rhizosphere.IMPORTANCE Diseases associated with fungal root invasion cause a significant loss of fruit tree production worldwide. The bacterium Pseudomonas pseudoalcaligenes AVO110 controls avocado white root rot disease caused by Rosellinia necatrix by using mechanisms involving competition for nutrients and niches. Here, a functional genomics approach was conducted to identify the bacterial traits involved in the interaction with this fungal pathogen. Our results contribute to a better understanding of the multitrophic interactions established among bacterial biocontrol agents, the plant rhizosphere, and the mycelia of soilborne pathogens.

Some facts about the respiratory enzymes of Pseudomonas pseudoalcaligenes KF707 recently renamed as Pseudomonas furukawaii sp. nov., type strain KF707.

Kimura and co-workers (Kimura N et al. Int J Syst Evol Microbiol 2018;68:1429-1435) recently proposed renaming the obligate aerobe Pseudomonas pseudoalcaligenes KF707 as Pseudomonas furukawiisp. nov. type strain KF707. Since the first quasi-complete genome sequence of KF707 was reported in 2012 (accession number: PRJNA83639) numerous reports on chemotaxis and function/composition of the respiratory redox chain of KF707 have been published, demonstrating that KF707 contains three cheA genes for aerobic motility, four cytochrome oxidases of c(c)aa3- and cbb3-type and one bd-type quinol oxidase. With this background in mind, it has been quite a surprise to read within Table 1 of the paper by Kimura et al. that strain KF707 is phenotypically characterized as cytochrome oxidase-negative. Further, Table 1 also reports that KF707 is ß-galactosidase-positive, an affirmation that is not consistent with results documented in the current literature. In this present 'Letter to the Editor' we show that Kimura et al. have contradicted themselves and provided inaccurate information in respect to the respiratory phenotypic features of P. furukawii. Based on this, an official corrigendum is requested since the publication, as it is, blurs the credibility of the International Journal of Systematic and Evolutionary Microbiology.

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.

Pseudomonas pseudoalcaligenes KF707 grown with biphenyl expresses a cytochrome caa3 oxidase that uses cytochrome c4 as electron donor.

Combining peroxidase activity-based heme staining (TMBZ/SDS/PAGE) with mass spectrometry analyses (Nano LC-MS/MS) of protein extracts from wild-type and appropriate mutants, we provide evidence that the polychlorinated biphenyl degrader Pseudomonas pseudoalcaligenes KF707 primarily expresses a caa3 -type cytochrome c oxidase (caa3 -Cox) using cytochrome (cyt) c4 as an electron donor in cells grown with biphenyl versus glucose as the sole carbon source. Homology modeling of KF707 caa3 -Cox using the three-dimensional structure of that from Thermus thermophilus highlights multiple similarities and differences between the proton channels in subunit I of the aa3 - and caa3 -Cox of Paracoccus and Thermus spp., respectively. To our knowledge, this is the first report demonstrating the presence of a caa3 -Cox using cyt c4 as an electron donor in a Pseudomonas species.