A phage tubulin assembles dynamic filaments by an atypical mechanism to center viral DNA within the host cell.

Tubulins are essential for the reproduction of many eukaryotic viruses, but historically, bacteriophage were assumed not to require a cytoskeleton. Here, we identify a tubulin-like protein, PhuZ, from bacteriophage 201φ2-1 and show that it forms filaments in vivo and in vitro. The PhuZ structure has a conserved tubulin fold, with an unusual, extended C terminus that we demonstrate to be critical for polymerization in vitro and in vivo. Longitudinal packing in the crystal lattice mimics packing observed by EM of in-vitro-formed filaments, indicating how interactions between the C terminus and the following monomer drive polymerization. PhuZ forms a filamentous array that is required for positioning phage DNA within the bacterial cell. Correct positioning to the cell center and optimal phage reproduction only occur when the PhuZ filament is dynamic. Thus, we show that PhuZ assembles a spindle-like array that functions analogously to the microtubule-based spindles of eukaryotes.

Lipopeptide-mediated bacterial interaction enables cooperative predator defense.

Bacteria are inherently social organisms whose actions should ideally be studied within an interactive ecological context. We show that the exchange and modification of natural products enables two unrelated bacteria to defend themselves against a common predator. Amoebal predation is a major cause of death in soil bacteria and thus it exerts a strong selective pressure to evolve defensive strategies. A systematic analysis of binary combinations of coisolated bacteria revealed strains that were individually susceptible to predation but together killed their predator. This cooperative defense relies on a Pseudomonas species producing syringafactin, a lipopeptide, which induces the production of peptidases in a Paenibacillus strain. These peptidases then degrade the innocuous syringafactin into compounds, which kill the predator. A combination of bioprospecting, coculture experiments, genome modification, and transcriptomics unravel this novel natural product-based defense strategy.

Analysis of single-cell microbial mass spectra profiles from single-particle aerosol mass spectrometry.

RATIONALE: Single-particle aerosol mass spectrometry is a practical method for studying microbial aerosols. However, the related mass spectral characteristics of single-cell microorganisms have not yet been studied systematically; hence, further investigations are necessary. METHODS: Different microbial cells were grown and directly aerosolized in the laboratory. These aerosols were then drawn into a single-particle mass spectrometer platform, and single-cell mass spectra profiles were obtained in real-time. The biological characteristics, ion variation trends, and microbial types were analyzed with either laser pulse energy or laser fluence. RESULTS: The single-particle mass spectra contained prominent peaks that could be attributed to the presence of biological matter, such as organic phosphate and nitrogen, amino acids, and spore-associated calcium complexes. Limited types of average mass spectral patterns were present, and a significant correlation was found between the ion intensity trend (presence and absence of peaks) and laser ionization energy (expressed by the total positive ion intensity). Although a single spectral data point does not contain all the peaks of the average spectrum, it covers most of the characteristic peaks and could be identified using a machine learning algorithm. After the analysis of single-particle mass spectra, we found that using multi-group features (e.g., peak intensity ratio of m/z +47 and +41, peak intensity ratio of 59 N(CH3 )3 + and 74 N(CH3 )4 + , and 12 peak variables) led to an identification accuracy of approximately 92.4% with the random forest algorithm. CONCLUSIONS: The results indicate that single-cell mass spectral profiles can be used to distinguish microbial aerosols and further illustrate their origin in a laboratory setting.

Sensitivity enhancement of the SPR biosensor for Pseudomonas bacterial detection employing a silicon-barium titanate structure.

A novel, to the best of our knowledge, surface plasmon resonance (SPR) sensor, employing a silicon-barium titanate structure for Pseudomonas bacterial detection, is designed. Three bacterial attachments operate as a protective layer for the detection process with refractive indices (RI) of 1.437, 1.49368, and 1.5265. Performance analysis shows a sensitivity (S) of 155, 168, and 370°/RIU at RI of 1.5265 for Structures 1, 2, and 3, respectively. Additionally, the proposed sensor (Structure 3) accomplishes a magnified figure of merit (FOM) of 86.43 and quality factor of 86.65 at the RI of 1.5265. Finally, the proposed sensor's performance is compared with that of the existing sensors, thus demonstrating a heightened S and FOM.

Effect of Pseudomonas moorei KB4 Cells' Immobilisation on Their Degradation Potential and Tolerance towards Paracetamol.

Pseudomonas moorei KB4 is capable of degrading paracetamol, but high concentrations of this drug may cause an accumulation of toxic metabolites. It is known that immobilisation can have a protective effect on bacterial cells; therefore, the toxicity and degradation rate of paracetamol by the immobilised strain KB4 were assessed. Strain KB4 was immobilised on a plant sponge. A toxicity assessment was performed by measuring the concentration of ATP using the colony-forming unit (CFU) method. The kinetic parameters of paracetamol degradation were estimated using the Hill equation. Toxicity analysis showed a protective effect of the carrier at low concentrations of paracetamol. Moreover, a pronounced phenomenon of hormesis was observed in the immobilised systems. The obtained kinetic parameters and the course of the kinetic curves clearly indicate a decrease in the degradation activity of cells after their immobilisation. There was a delay in degradation in the systems with free cells without glucose and immobilised cells with glucose. However, it was demonstrated that the immobilised systems can degrade at least ten succeeding cycles of 20 mg/L paracetamol degradation. The obtained results indicate that the immobilised strain may become a useful tool in the process of paracetamol degradation.

Inactivation and Membrane Damage Mechanism of Slightly Acidic Electrolyzed Water on Pseudomonas deceptionensis CM2.

Pseudomonas is considered as the specific spoilage bacteria in meat and meat products. The purpose of this study was to evaluate the inactivation efficiency and mechanisms of slightly acidic electrolyzed water (SAEW) against Pseudomonas deceptionensis CM2, a strain isolated from spoiling chicken breast. SAEW caused time-dependent inactivation of P. deceptionensis CM2 cells. After exposure to SAEW (pH 5.9, oxidation-reduction potential of 945 mV, and 64 mg/L of available chlorine concentration) for 60 s, the bacterial populations were reduced by 5.14 log reduction from the initial load of 10.2 log10 CFU/mL. Morphological changes in P. deceptionensis CM2 cells were clearly observed through field emission-scanning electron microscopy as a consequence of SAEW treatment. SAEW treatment also resulted in significant increases in the extracellular proteins and nucleic acids, and the fluorescence intensities of propidium iodide and n-phenyl-1-napthylamine in P. deceptionensis CM2 cells, suggesting the disruption of cytoplasmic and outer membrane integrity. These findings show that SAEW is a promising antimicrobial agent.

Denitrifier Method for Nitrite Removal in Electrochemical Analysis of the Electron Accepting Capacity of Humic Substances.

Humic substances (HSs) are important electron acceptors and donors in soils and aquifers. The coupling of anoxic nitrogen (N) cycling to the function of HSs as a redox battery, however, remains poorly understood. Mediated electrochemical analysis is an emerging tool to determine the redox properties (i.e., electron donating capacity (EDC), electron accepting capacity (EAC), and redox state) of HS. However, the presence of nitrite (NO2-), a central intermediate of the N-cycle, interferes with the electrochemical determination of the EAC. To eliminate this interference, we developed a bioassay to remove nitrite in HS samples using the denitrifying bacterium Pseudomonas nitroreducens. Cell suspensions of P. nitroreducens completely removed NO2- at various concentrations (1, 2, and 5 mM) from humic acid samples (1 g HA/L) of different redox states. As P. nitroreducens is not able to exchange electrons with dissolved humic acids, the procedure allows an accurate and reliable determination of the EAC of humic acid samples. The proposed method thus opens new perspectives in biogeochemistry to study interactions between HSs and N cycling.

Pseudomonas atacamensis sp. nov., isolated from the rhizosphere of desert bloom plant in the region of Atacama, Chile.

The bacterial strain M7D1T was isolated from samples of the rhizosphere of desert bloom plants on the Atacama region located in northern Chile as part of a study intended to isolate nitrifying bacteria in this adverse environment. It was previously identified as belonging to the Pseudomonas fluorescens group. In this study, the phylogenetic analysis of the 16s RNA, gyrA, rpoB and rpoD genes confirmed that this strain belongs to this group, especially Sub Group (SG) Koreensis, but it represents a potential new species. Additionally, the average nucleotide identity confirmed this as the highest identity value (0.92) with Pseudomonas moraviensis LMG 24280, which is lower than the 0.94 threshold established to classify two strains within the same species. The strain M7D1T shared a similar fatty acids methyl ester profile than the type strains of other Pseudomonas spp. previously described. Furthermore, it can be differentiated phenotypically from other related species of SG P. koreensis. Based on these results, the existence of a new species of Pseudomonas is demonstrated, for which the name Pseudomonas atacamensis is proposed. This strain presented a set of genes associated with plant growth-promoting rhizobacteria and it is a good candidate to be used for recovery of contaminated soils. However, more studies are required to demonstrate whether this bacterium is non-pathogenic, can survive in the presence of toxic compounds and promote growth or help to the stress management of plants.

Characterization of methyl-accepting chemotaxis proteins (MCPs) for amino acids in plant-growth-promoting rhizobacterium Pseudomonas protegens CHA0 and enhancement of amino acid chemotaxis by MCP genes overexpression.

Pseudomonas protegens CHA0, known as plant-growth-promoting rhizobacterium, showed positive chemotactic responses toward proteinaceous L-amino acids. Genomic analysis revealed that P. protegens CHA0 possesses four putative chemoreceptors for amino acids (designated CtaA, CtaB, CtaC, and CtaD, respectively). Pseudomonas aeruginosa PCT2, a mutant defective in chemotaxis to amino acids, harboring a plasmid containing each of ctaA, ctaB, ctaC, and ctaD showed chemotactic responses to 20, 4, 4, and 11 types of amino acids, respectively. To enhance chemotaxis toward amino acids, we introduced the plasmids containing ctaA, ctaB, ctaC, or ctaD into P. protegens CHA0. By overexpression of the genes, we succeeded in enhancing chemotaxis toward more than half of the tested ligands. However, unexpectedly, the P. protegens CHA0 transformants showed unchanged or decreased responses to some amino acids when compared to wild-type CHA0. We speculate that alternation of expression of a chemoreceptor may affect the abundance of other chemoreceptors. ABBREVIATIONS: cDNA: complementary DNA; LBD: ligand-binding domain; MCP: methyl-accepting chemotaxis protein; PDC: PhoQ/DcuS/CitA; PGPR: plant-growth-promoting rhizobacteria; qRT-PCR: quantitative reverse transcription PCR.

Exploring encapsulation strategies as a protective mechanism to avoid amensalism in mixed populations of Pseudomonas taetrolens and Lactobacillus casei.

Pseudomonas taetrolens constitutes an efficient platform for the biosynthesis of lactobionic acid, a potentially prebiotic compound. Unfortunately, an amensalistic interaction has been demonstrated between P. taetrolens and probiotic lactic acid bacteria (LAB), characterized by the competitive exclusion of P. taetrolens, hindering the in situ production of fermented dairy products with synbiotic properties. In the present research, encapsulation was explored as a barrier to the diffusion of the antimicrobial metabolites generated by LAB. Mixed fermentations involving P. taetrolens LMG 2336 and Lactobacillus casei CECT 475 were cultivated, entrapping both microorganisms alternately. Alginate, alginate/starch and carboxymethyl cellulose/k-carrageenan were tested as encapsulating agents. The immobilization of L. casei in 2% alginate/2% starch beads was found to be the best strategy, improving the production of lactobionic acid by 182% with respect to co-cultures with free cells. This study proves the potential of LAB encapsulation for the protection of sensitive strains in mixed food fermentations.

High intensity light pulses to reduce microbial load in fresh cheese.

The present study focused on the utilisation of High Intensity Light Pulses (HILP) treatment to preserve mozzarella cheese. First, the susceptibility of Pseudomonas fluorescens and Enterobacteriaceae to HILP (fluences from 0·39 to 28·0 J/cm2) in a transparent liquid was evaluated (in-vitro tests). Afterwards, the effects on inoculated mozzarella cheese were also assessed. Then untreated (Control) and HILP treated samples were packaged and stored at 10 °C for 2 weeks. Enterobacteriaceae, Pseudomonas spp. and pH were monitored during storage. In a transparent liquid (in-vitro tests) there was a significant microbial inactivation just with 2 s of treatment. On the inoculated cheese a relevant microbial reduction of about 1 log cycle was observed, according to the exposure to the treatments. For Pseudomonas spp. in particular, in the treated samples, the microbiological acceptability limit (106 cfu/g) was never reached after 2 weeks of refrigerated storage. To sum up, the efficacy of this treatment is very interesting because a microbial reduction was observed in treated samples. HILP treatment is able to control the microbial growth and may be considered a promising way to decontaminate the surface of mozzarella cheese.

Characterization of a cold-adapted esterase and mutants from a psychotolerant Pseudomonas sp. strain.

A cold-adapted esterase-producing strain named T1-39 was isolated from Glacier No. 1, Tianshan, People's Republic of China and identified as Pseudomonas sp. from 16S rRNA sequence analysis. The esterase (EstT1-39) secreted by this strain preferentially hydrolyzed esters of glycerol with short- and medium-chain fatty acids. Mutants of T1-39 were generated by the atmospheric and room temperature plasma method and screened for enhanced esterase activity. Among all the mutants, strain TB11 had 4.45-fold higher esterase productivity than T1-39, with high genetic stability over 10 generations of continuous cultivation. Maximum activity of EstT1-39 and EstTB11 was observed at 30 ℃, pH 9.0 and 25 ℃, pH 8.5, respectively. EstTB11 was thermally more stable (50 ℃ for 1 H) and active over a broader pH range than EstT1-39. EstTB11 also retained 38% of its maximal activity at 0 ℃ and was found to be able to hydrolyze milk fats into short- and medium-chain fatty acids at 4 ℃. The characteristics of EstT1-39 made it a cold-adapted enzyme and the EstTB11 from the mutant, with its higher activity at lower temperatures, may be suitable for the production of aromas and flavors in the dairy industry.

Stereoselective biotransformation of phenylglycine nitrile by heterogeneous biocatalyst based on immobilized bacterial cells and enzyme preparation.

We studied the effect of a heterogeneous environment on the stereoselectivity of transformation of racemic phenylglycine nitrile. Immobilized biocatalysts were prepared by adhesion of Pseudomonas fluorescens C2 cells on carbon-containing supports and covalent crosslinking of nitrile hydratase and amidase of Rhodococcus rhodochrous 4-1 to activated chitosan as well as by the method of cross-linked aggregates. At a reaction duration of 20 h, the ratio of phenylglycine stereoisomers changes depending on the presence of support in medium. The highest optical purity of the product (enantiomeric excess of L-phenylglycine solution, 98%) is achieved when enzyme aggregates of nitrile hydratase and amidase cross-linked with 0.1% glutaraldehyde are used as a biocatalyst.

Cellular Viscosity in Prokaryotes and Thermal Stability of Low Molecular Weight Biomolecules.

Some low molecular weight biomolecules, i.e., NAD(P)H, are unstable at high temperatures. The use of these biomolecules by thermophilic microorganisms has been scarcely analyzed. Herein, NADH stability has been studied at different temperatures and viscosities. NADH decay increased at increasing temperatures. At increasing viscosities, NADH decay rates decreased. Thus, maintaining relatively high cellular viscosity in cells could result in increased stability of low molecular weight biomolecules (i.e., NADH) at high temperatures, unlike what was previously deduced from studies in diluted water solutions. Cellular viscosity was determined using a fluorescent molecular rotor in various prokaryotes covering the range from 10 to 100°C. Some mesophiles showed the capability of changing cellular viscosity depending on growth temperature. Thermophiles and extreme thermophiles presented a relatively high cellular viscosity, suggesting this strategy as a reasonable mechanism to thrive under these high temperatures. Results substantiate the capability of thermophiles and extreme thermophiles (growth range 50-80°C) to stabilize and use generally considered unstable, universal low molecular weight biomolecules. In addition, this study represents a first report, to our knowledge, on cellular viscosity measurements in prokaryotes and it shows the dependency of prokaryotic cellular viscosity on species and growth temperature.

Living on the edge: emergence of spontaneous gac mutations in Pseudomonas protegens during swarming motility.

Swarming motility is a flagella-driven multicellular behaviour that allows bacteria to colonize new niches and escape competition. Here, we investigated the evolution of specific mutations in the GacS/GacA two-component regulatory system in swarming colonies of Pseudomonas protegens Pf-5. Experimental evolution assays showed that repeated rounds of swarming by wildtype Pf-5 drives the accumulation of gacS/gacA spontaneous mutants on the swarming edge. These mutants cannot swarm on their own because they lack production of the biosurfactant orfamide A, but they do co-swarm with orfamide-producing wildtype Pf-5. These co-swarming assays further demonstrated that ΔgacA mutant cells indeed predominate on the edge and that initial ΔgacA:wildtype Pf-5 ratios of at least 2:1 lead to a collapse of the swarming colony. Subsequent whole-genome transcriptome analyses revealed that genes associated with motility, resource acquisition, chemotaxis and efflux were significantly upregulated in ΔgacA mutant on swarming medium. Moreover, transmission electron microscopy showed that ΔgacA mutant cells were longer and more flagellated than wildtype cells, which may explain their predominance on the swarming edge. We postulate that adaptive evolution through point mutations is a common feature of range-expanding microbial populations and that the putative fitness benefits of these mutations during dispersal of bacteria into new territories are frequency-dependent.

Rapid Identification of Pseudomonas spp. via Raman Spectroscopy Using Pyoverdine as Capture Probe.

Pyoverdine is a substance which is excreted by fluorescent pseudomonads in order to scavenge iron from their environment. Due to specific receptors of the bacterial cell wall, the iron loaded pyoverdine molecules are recognized and transported into the cell. This process can be exploited for developing efficient isolation and enrichment strategies for members of the Pseudomonas genus, which are capable of colonizing various environments and also include human pathogens like P. aeruginosa and the less virulent P. fluorescens. A significant advantage over antibody based systems is the fact that siderophores like pyoverdine can be considered as "immutable ligands," since the probability for mutations within the siderophore uptake systems of bacteria is very low. While each species of Pseudomonas usually produces structurally unique pyoverdines, which can be utilized only by the producer strain, cross reactivity does occur. In order to achieve a reliable identification of the captured pathogens, further investigations of the isolated cells are necessary. In this proof of concept study, we combine the advantages of an isolation strategy relying on "immutable ligands" with the high specificity and speed of Raman microspectroscopy. In order to isolate the bacterial cells, pyoverdine was immobilized covalently on planar aluminum chip substrates. After capturing, single cell Raman spectra of the isolated species were acquired. Due to the specific spectroscopic fingerprint of each species, the bacteria can be identified. This approach allows a very rapid detection of potential pathogens, since time-consuming culturing steps are unnecessary. We could prove that pyoverdine based isolation of bacteria is fully Raman compatible and further investigated the capability of this approach by isolating and identifying P. aeruginosa and P. fluorescens from tap water samples, which are both opportunistic pathogens and can pose a threat for immunocompromised patients.

Cryo-electron tomography of plunge-frozen whole bacteria and vitreous sections to analyze the recently described bacterial cytoplasmic structure, the Stack.

Cryo-electron tomography (CET) of plunge-frozen whole bacteria and vitreous sections (CETOVIS) were used to revise and expand the structural knowledge of the "Stack", a recently described cytoplasmic structure in the Antarctic bacterium Pseudomonas deceptionensis M1(T). The advantages of both techniques can be complementarily combined to obtain more reliable insights into cells and their components with three-dimensional imaging at different resolutions. Cryo-electron microscopy (Cryo-EM) and CET of frozen-hydrated P. deceptionensis M1(T) cells confirmed that Stacks are found at different locations within the cell cytoplasm, in variable number, separately or grouped together, very close to the plasma membrane (PM) and oriented at different angles (from 35° to 90°) to the PM, thus establishing that they were not artifacts of the previous sample preparation methods. CET of plunge-frozen whole bacteria and vitreous sections verified that each Stack consisted of a pile of oval disc-like subunits, each disc being surrounded by a lipid bilayer membrane and separated from each other by a constant distance with a mean value of 5.2±1.3nm. FM4-64 staining and confocal microscopy corroborated the lipid nature of the membrane of the Stacked discs. Stacks did not appear to be invaginations of the PM because no continuity between both membranes was visible when whole bacteria were analyzed. We are still far from deciphering the function of these new structures, but a first experimental attempt links the Stacks with a given phase of the cell replication process.

Simultaneous removal of harmful algal blooms and microcystins using microorganism- and chitosan-modified local soil.

Cyanobacterial harmful algal blooms (cyano-HAB) and microcystins (MCs) can cause a potential threat to public health. Here, a method for simultaneous removal of cyano-HAB and MCs was developed using chitosan-modified local soil (MLS) flocculation plus microorganism-modified soil capping. The experiment was conducted in simulated columns containing algal water collected from Lake Taihu (China). More than 90% of algal cells and intracellular MCs were flocculated and removed from water using chitosan-MLS and the sunken flocs were treated by different capping materials including Pseudomonas sp. An18 modified local soil. During 40 days of incubation, dissolved MC-LR and MC-RR showed 10-fold increase in the flocculation-only system. The increase of MC-LR and MC-RR in water was reduced by 30 and 70% in soil capping treatments; however, the total content of MCs in the sediment-water column remained similar to that in the control and flocculation only systems. In contrast, both dissolved MCs and total MCs were reduced by 90% in Pseudomonas sp. An18 modified soil capping treatment. The high performance of toxin decomposition was due to the combined effects of flocculation and MC-degrading bacteria that embedded in the capping material, which prevents dilution of bacteria biomass, concentrates algal cells, confines released toxins, and enhances toxin biodegradation.

Biotransformation and chemotaxis of 4-chloro-2-nitrophenol by Pseudomonas sp. JHN.

Pseudomonas sp. JHN decolourized and biotransformed 4-chloro-2-nitrophenol (4C2NP) in the presence of additional carbon source. The effect of the various concentrations of the 4C2NP was studied on the decolourization of 4C2NP by Pseudomonas sp. JHN. It was observed that strain JHN decolourized and biotransformed 4C2NP up to concentration of 0.6 mM. Gas chromatography and gas chromatography-mass spectrometry detected 5-chloro-2-methylbenzoxazole as a major metabolite of the co-metabolism of 4C2NP. Furthermore, strain JHN exhibits positive chemotaxis toward 4C2NP based on the drop plate and capillary assays. This is the first report of the chemotaxis toward 4C2NP by any bacterium.

The acute transcriptional response of the coral Acropora millepora to immune challenge: expression of GiMAP/IAN genes links the innate immune responses of corals with those of mammals and plants.

BACKGROUND: As a step towards understanding coral immunity we present the first whole transcriptome analysis of the acute responses of Acropora millepora to challenge with the bacterial cell wall derivative MDP and the viral mimic poly I:C, defined immunogens provoking distinct but well characterised responses in higher animals. RESULTS: These experiments reveal similarities with the responses both of arthropods and mammals, as well as coral-specific effects. The most surprising finding was that MDP specifically induced three members of the GiMAP gene family, which has been implicated in immunity in mammals but is absent from Drosophila and Caenorhabditis. Like their mammalian homologs, GiMAP genes are arranged in a tandem cluster in the coral genome. CONCLUSIONS: A phylogenomic survey of this gene family implies ancient origins, multiple independent losses and lineage-specific expansions during animal evolution. Whilst functional convergence cannot be ruled out, GiMAP expression in corals may reflect an ancestral role in immunity, perhaps in phagolysosomal processing.