Inhibitory effects of Pseudomonas sp. W112 on cadmium accumulation in wheat grains: Reduced the bioavailability in soil and enhanced the interception by plant organs.

Microorganisms play an important role in heavy metal bioremediation and soil fertility. The effects of soil inoculation with Pseudomonas sp. W112 on Cd accumulation in wheat were investigated by analyzing the transport, subcellular distribution and speciation of Cd in the soil and plants. Pseudomonas sp. W112 application significantly decreased Cd content in the roots, internode and grains by 10.2%, 29.5% and 33.0%, respectively, and decreased Cd transfer from the basal nodes to internodes by 63.5%. Treatment with strain W112 decreased the inorganic and water-soluble Cd content in the roots and increased the proportion of residual Cd in both the roots and basal nodes. At the subcellular level, the Cd content in the root cell wall and basal node cytosol increased by 19.6% and 61.8%, respectively, indicating that strain W112 improved the ability of the root cell wall and basal node cytosol to fix Cd. In the rhizosphere soil, strain W112 effectively colonized and significantly decreased the exchangeable Cd, carbonate-bound Cd and iron-manganese oxide-bound Cd content by 43.5%, 27.3% and 17.6%, respectively, while it increased the proportion of residual Cd by up to 65.2%. Moreover, a 3.1% and 23.5% increase in the pH and inorganic nitrogen content in the rhizosphere soil, respectively, was recorded. Similarly, soil bacterial community sequencing revealed that inoculating with strain W112 increased the abundance of Pseudomonas, Thauera and Azoarcus, which are associated with inorganic nitrogen metabolism, and decreased that of Acidobacteria, which is indicative of soil alkalinization. Hence, root application of Pseudomonas sp. W112 improved soil nitrogen availability and inhibited Cd accumulation in the wheat grains in a two-stage process: by reducing the Cd availability in the rhizosphere soil and by improving Cd interception and fixation in the wheat roots and basal nodes. Pseudomonas sp. W112 may be a suitable bioremediation agent for restoring Cd-contaminated wheat fields.

Molecular mechanism of siderophore regulation by the Pseudomonas aeruginosa BfmRS two-component system in response to osmotic stress.

Pseudomonas aeruginosa, a common nosocomial pathogen, relies on siderophores to acquire iron, crucial for its survival in various environments and during host infections. However, understanding the molecular mechanisms of siderophore regulation remains incomplete. In this study, we found that the BfmRS two-component system, previously associated with biofilm formation and quorum sensing, is essential for siderophore regulation under high osmolality stress. Activated BfmR directly bound to the promoter regions of pvd, fpv, and femARI gene clusters, thereby activating their transcription and promoting siderophore production. Subsequent proteomic and phenotypic analyses confirmed that deletion of BfmRS reduces siderophore-related proteins and impairs bacterial survival in iron-deficient conditions. Furthermore, phylogenetic analysis demonstrated the high conservation of the BfmRS system across Pseudomonas species, functional evidences also indicated that BfmR homologues from Pseudomonas putida KT2440 and Pseudomonas sp. MRSN12121 could bind to the promoter regions of key siderophore genes and osmolality-mediated increases in siderophore production were observed. This work illuminates a novel signaling pathway for siderophore regulation and enhances our understanding of siderophore-mediated bacterial interactions and community establishment.

Pyoluteorin regulates the biosynthesis of 2,4-DAPG through the TetR family transcription factor PhlH in Pseudomonas protegens Pf-5.

Soil and rhizosphere bacteria act as a rich source of secondary metabolites, effectively fighting against a diverse array of pathogens. Certain Pseudomonas species harbor biosynthetic gene clusters for producing both pyoluteorin and 2,4-diacetylphloroglucinol (2,4-DAPG), which are polyketides that exhibit highly similar antimicrobial spectrum against bacteria and fungi or oomycete. A complex cross talk exists between pyoluteorin and 2,4-DAPG biosynthesis, and production of 2,4-DAPG was strongly repressed by pyoluteorin, yet the underlying mechanism is still elusive. In this study, we find that the TetR family transcription factor PhlH is involved in the cross talk between pyoluteorin and 2,4-DAPG biosynthesis. PhlH binds to a palindromic sequence within the promoter of phlG (PphlG), which encodes a C-C bond hydrolase responsible for degrading 2,4-DAPG. As a signaling molecule, pyoluteorin disrupts the PhlH-PphlG complex by binding to PhlH, leading to decreased levels of 2,4-DAPG. Proteomics data suggest that pyoluteorin regulates multiple physiological processes including fatty acid biosynthesis and transportation of taurine, siderophore, and amino acids. Our work not only reveals a novel mechanism of cross talk between pyoluteorin and 2,4-DAPG biosynthesis, but also highlights pyoluteorin's role as a messenger in the complex communication network of Pseudomonas.IMPORTANCEAntibiosis serves as a crucial defense mechanism for microbes against invasive bacteria and resource competition. These bacteria typically orchestrate the production of multiple antibiotics in a coordinated fashion, wherein the synthesis of one antibiotic inhibits the generation of another. This strategic coordination allows the bacterium to focus its resources on producing the most advantageous antibiotic under specific circumstances. However, the underlying mechanisms of distinct antibiotic production in bacterial cells remain largely elusive. In this study, we reveal that the TetR family transcription factor PhlH detects the secondary metabolite pyoluteorin and mediates the cross talk between pyoluteorin and 2,4-DAPG biosynthesis in the biocontrol strain Pseudomonas protegens Pf-5. These findings hold promise for future research, potentially informing the manipulation of these systems to enhance the effectiveness of biocontrol agents.

Synergistic application of Pseudomonas strains and compost mitigates lead (Pb) stress in sunflower (Helianthus annuus L.) via improved nutrient uptake, antioxidant defense and physiology.

Lead (Pb) is one of the most dreadful non-essential elements whose toxicity has been well reported worldwide due to its interference with the major plant functions and its overall yield. Bioremediation techniques comprising the application of beneficial microorganisms have gained attention in recent times owing to their ecofriendly nature. Addition of organic matter to soil has been reported to stimulate microbial activities. Compost application improves soil structure and binds toxic contaminants due to its larger surface area and presence of functional groups. Furthermore, it stimulates soil microbial activities by acting as C-source. So, in current study, we investigated the individual and synergistic potential of two lead (Pb)-tolerant Pseudomonas strains alongwith compost (1% w/w) in sustaining sunflower growth under Pb contaminated soil conditions. Lead chloride (PbCl2) salt was used for raising desired Pb concentration (500 mg kg-1). Results revealed that Pb stress drastically affected all the measured attributes of sunflower plant, however joint application of rhizobacteria and compost counteracted these adverse effects. Among them, co-application of str-1 and compost proved to be significantly better than str-2, as its inoculation significantly improved shoot and root lengths (64 and 76%), leaf area and leaves plant-1 (95 and 166%), 100-achene weight (200%), no. of flowers plant-1 (138%), chl 'a', 'b' and carotenoid (86, 159 and 33%) contents in sunflower as compared to control treatments. Furthermore, inoculation of Pseudomonas fluorescens along with compost increased the NPK in achene (139, 200 and 165%), flavonoid and phenolic contents (258 and 185%) along with transpiration and photosynthetic rates (54 and 72%) in leaves as compared to control treatment under Pb contamination. In addition, Pb entry to roots, shoots and achene were significantly suppressed under by 87, 90 and 91% respectively due to integrated application of compost and str-1 as evident by maximum Pb-immobilization efficiency (97%) obtained in this treatment. Similarly, bioconcentration factors for roots, shoots and achene were found to be 0.58, 0.18 and 0.0055 with associated translocation factor (0.30), which also revealed phytostabilization of Pb under combined application of PGPR and compost. Since, phytoremediation of heavy metals under current scenario of increasing global population is inevitable, results of the current study concluded that tolerant PGPR species along with organic amendments such as compost can inhibit Pb uptake by sunflower and confer Pb tolerance via improved nutrient uptake, physiology, antioxidative defense and gas exchange.

Can xenobiotics support the growth of Mn(II)-oxidizing bacteria (MnOB)? A case of phenol-utilizing bacteria Pseudomonas sp. AN-1.

Biogenic manganese oxides (BioMnOx) produced by Mn(II)-oxidizing bacteria (MnOB) have garnered considerable attention for their exceptional adsorption and oxidation capabilities. However, previous studies have predominantly focused on the role of BioMnOx, neglecting substantial investigation into MnOB themselves. Meanwhile, whether the xenobiotics could support the growth of MnOB as the sole carbon source remains uncertain. In this study, we isolated a strain termed Pseudomonas sp. AN-1, capable of utilizing phenol as the sole carbon source. The degradation of phenol took precedence over the accumulation of BioMnOx. In the presence of 100 mg L-1 phenol and 100 µM Mn(II), phenol was entirely degraded within 20 h, while Mn(II) was completely oxidized within 30 h. However, at the higher phenol concentration (500 mg L-1), phenol degradation reduced to 32% and Mn(II) oxidation did not appear to occur. TOC determination confirmed the ability of strain AN-1 to mineralize phenol. Based on the genomic and proteomics studies, the Mn(II) oxidation and phenol mineralization mechanism of strain AN-1 was further confirmed. Proteome analysis revealed down-regulation of proteins associated with Mn(II) oxidation, including MnxG and McoA, with increasing phenol concentration. Notably, this study observed for the first time that the expression of Mn(II) oxidation proteins is modulated by the concentration of carbon sources. This work provides new insight into the interaction between xenobiotics and MnOB, thus revealing the complexity of biogeochemical cycles of Mn and C.

Taming Pseudomonas aeruginosa AM26 the barbarian: Targeting the PQS quorum sensing network using crude mandarin extract.

Pseudomonas aeruginosa, one of the most notorious organisms, causes fatal diseases like-, meningitis, pneumonia as well as worsens the prognosis of cystic fibrosis patients. It is also multi-drug resistant and resists a wide range of antibiotics. Attempts have been made to reduce its virulence/pathogenic potential using a number of organic compounds. For this purpose, the Quorum sensing (QS) system of P. aeruginosa was targeted, which regulates its virulence. Pseudomonas Quinolone System (PQS), one of the four quorum sensing systems, producing pyocyanin pigment was chosen. 2-heptyl-3-hydroxy-4-quinolone (HHQ) is a ligand which binds to PQS protein is responsible for pyocyanin pigment production. Attempts were made to find a compound analogous to HHQ which could bind to PQS active site and inhibit the pigment formation. In-silico analysis was performed to estimate possible interactions and to find/predict the possible PQS inhibitors.

The Role of Complement Component C3 in Protection Against Pseudomonas Pneumonia-Induced Lung Injury.

The complement system is a family of proteins that facilitate immune resistance by attacking microbes to decrease pathogen burden. As a result, deficiencies of certain complement proteins result in recurrent bacterial infections, and can also result in acute lung injury (ALI). We and others have shown that C3 is present in both immune and nonimmune cells, and modulates cellular functions such as metabolism, differentiation, cytokine production, and survival. Although the emerging roles of the complement system have implications for host responses to ALI, key questions remain vis-a-vis the lung epithelium. In this review, we summarize our recent article in which we reported that during Pseudomonas aeruginosa-induced ALI, lung epithelial cell-derived C3 operates independent of liver-derived C3. Specifically, we report the use of a combination of human cell culture systems and global as well as conditional knockout mouse models to demonstrate the centrality of lung epithelial cell-derived C3. We also summarize recent articles that have interrogated the role of intracellular and/or locally derived C3 in host defense. We propose that C3 is a highly attractive candidate for enhancing tissue resilience in lung injury as it facilitates the survival and function of the lung epithelium, a key cell type that promotes barrier function.

The Caenorhabditis elegans proteome response to two protective Pseudomonas symbionts.

The Caenorhabditis elegans natural microbiota isolates Pseudomonas lurida MYb11 and Pseudomonas fluorescens MYb115 protect the host against pathogens through distinct mechanisms. While P. lurida produces an antimicrobial compound and directly inhibits pathogen growth, P. fluorescens MYb115 protects the host without affecting pathogen growth. It is unknown how these two protective microbes affect host biological processes. We used a proteomics approach to elucidate the C. elegans response to MYb11 and MYb115. We found that both Pseudomonas isolates increase vitellogenin protein production in young adults, which confirms previous findings on the effect of microbiota on C. elegans reproductive timing. Moreover, the C. elegans responses to MYb11 and MYb115 exhibit common signatures with the response to other vitamin B12-producing bacteria, emphasizing the importance of vitamin B12 in C. elegans-microbe metabolic interactions. We further analyzed signatures in the C. elegans response specific to MYb11 or MYb115. We provide evidence for distinct modifications in lipid metabolism by both symbiotic microbes. We could identify the activation of host-pathogen defense responses as an MYb11-specific proteome signature and provide evidence that the intermediate filament protein IFB-2 is required for MYb115-mediated protection. These results indicate that MYb11 not only produces an antimicrobial compound but also activates host antimicrobial defenses, which together might increase resistance to infection. In contrast, MYb115 affects host processes such as lipid metabolism and cytoskeleton dynamics, which might increase host tolerance to infection. Overall, this study pinpoints proteins of interest that form the basis for additional exploration into the mechanisms underlying C. elegans microbiota-mediated protection from pathogen infection and other microbiota-mediated traits.IMPORTANCESymbiotic bacteria can defend their host against pathogen infection. While some protective symbionts directly interact with pathogenic bacteria, other protective symbionts elicit a response in the host that improves its own pathogen defenses. To better understand how a host responds to protective symbionts, we examined which host proteins are affected by two protective Pseudomonas bacteria in the model nematode Caenorhabditis elegans. We found that the C. elegans response to its protective symbionts is manifold, which was reflected in changes in proteins that are involved in metabolism, the immune system, and cell structure. This study provides a foundation for exploring the contribution of the host response to symbiont-mediated protection from pathogen infection.

Optimization of Growth Conditions for Chlorpyrifos-Degrading Bacteria in Farm Soils in Nakuru County, Kenya.

Chlorpyrifos (CP) is a chlorinated organophosphate pesticide. In Kenya, it is commonly used as an acaricide, particularly in dairy farming, leading to soil and water contamination. The study is aimed at isolating bacteria with CP-degrading potential and optimizing their growth conditions, including temperature, pH, and CP concentration. The enrichment culture technique was used, with minimal salt medium (MSM) supplemented with commercial grade CP. A multilevel factorial design was used to investigate the interactions of temperature, pH, and CP concentration. According to the findings, seven bacterial strains with potential to degrade CP were characterized and identified as Alcaligenes faecalis, Bacillus weihenstephanensis, Bacillus toyonensis, Alcaligenes sp. strain SCAU23, Pseudomonas sp. strain PB845W, Brevundimonas diminuta, and uncultured bacterium clone 99. Growth and biodegradation of bacteria differed significantly among the isolates across pH value, temperature, and concentrations (P ≤ 0.05). The optimum conditions for growth were pH 7, temperature of 25°C, and 25mg/l chlorpyrifos concentration, while optimum degradation conditions were pH 5, temp 25°C, and CP conc. 25mg/l. The Pearson correlation between optimum growth and degradation showed a weak positive relationship (R = 0.1144) for pH and strong positive relationship for temperature and concentration of chlorpyrifos. Other than pH, the study shows that there could be other cofactors facilitating the chlorpyrifos degradation process. The findings show that an efficient consortium, at 25°C and pH 5, can include Bacillus toyonensis 20SBZ2B and Alcaligenes sp. SCAU23 as they showed high optical density (OD) values under these conditions. These results indicate the potential for these bacteria to be employed in chlorpyrifos-contaminated ecosystem detoxification efforts upon manipulation of natural growth conditions. The findings of this study offer a potential foundation for future research into the reconstitution of a consortium. Based on the optimum conditions identified, the isolated bacterial strains could be further developed into a consortium to effectively degrade CP in both laboratory and field conditions. Dairy farmers can utilize the isolated strains and the consortia to decontaminate farm soils.

Pseudomonas-associated bacteria play a key role in obtaining nutrition from bamboo for the giant panda (Ailuropoda melanoleuca).

Gut microbiota plays a vital role in obtaining nutrition from bamboo for giant pandas. However, low cellulase activity has been observed in the panda's gut. Besides, no specific pathway has been implicated in lignin digestion by gut microbiota of pandas. Therefore, the mechanism by which they obtain nutrients is still controversial. It is necessary to elucidate the precise pathways employed by gut microbiota of pandas to degrade lignin. Here, the metabolic pathways for lignin degradation in pandas were explored by comparing 209 metagenomic sequencing data from wild species with different feeding habits. Lignin degradation central pathways, including beta-ketoadipate and homogentisate pathway, were enriched in the gut of wild bamboo-eating pandas. The gut microbiome of wild bamboo-eating specialists was enriched with genes from pathways implicated in degrading ferulate and p-coumarate into acetyl-CoA and succinyl-CoA, which can potentially provide the raw materials for metabolism in pandas. Specifically, Pseudomonas, as the most dominant gut bacteria genus, was found to be the main bacteria to provide genes involved in lignin or lignin derivative degradation. Herein, three Pseudomonas-associated strains isolated from the feces of wild pandas showed the laccase, lignin peroxidase, and manganese peroxidase activity and extracellular lignin degradation ability in vitro. A potential mechanism for pandas to obtain nutrition from bamboo was proposed based on the results. This study provides novel insights into the adaptive evolution of pandas from the perspective of lignin metabolism. IMPORTANCE: Although giant pandas only feed on bamboo, the mechanism of lignin digestion in pandas is unclear. Here, the metabolic pathways for lignin degradation in wild pandas were explored by comparing gut metagenomic from species with different feeding habits. Results showed that lignin degradation central pathways, including beta-ketoadipate and homogentisate pathway, were enriched in the gut of wild bamboo-eating pandas. Genes from pathways involved in degrading ferulate and p-coumarate via beta-ketoadipate pathway were also enriched in bamboo-eating pandas. The final products of the above process, such as acetyl-CoA, can potentially provide the raw materials for metabolism in pandas. Specifically, Pseudomonas, as the most dominant gut bacteria genus, mainly provides genes involved in lignin degradation. Herein, Pseudomonas-associated strains isolated from the feces of pandas could degrade extracellular lignin. These findings suggest that gut microbiome of pandas is crucial in obtaining nutrition from lignin via Pseudomonas, as the main lignin-degrading bacteria.

Engineering styrene biosynthesis: designing a functional trans-cinnamic acid decarboxylase in Pseudomonas.

We are interested in converting second generation feedstocks into styrene, a valuable chemical compound, using the solvent-tolerant Pseudomonas putida DOT-T1E as a chassis. Styrene biosynthesis takes place from L-phenylalanine in two steps: firstly, L-phenylalanine is converted into trans-cinnamic acid (tCA) by PAL enzymes and secondly, a decarboxylase yields styrene. This study focuses on designing and synthesizing a functional trans-cinnamic acid decarboxylase in Pseudomonas putida. To achieve this, we utilized the "wholesale" method, involving deriving two consensus sequences from multi-alignments of homologous yeast ferulate decarboxylase FDC1 sequences with > 60% and > 50% identity, respectively. These consensus sequences were used to design Pseudomonas codon-optimized genes named psc1 and psd1 and assays were conducted to test the activity in P. putida. Our results show that the PSC1 enzyme effectively decarboxylates tCA into styrene, whilst the PSD1 enzyme does not. The optimal conditions for the PSC1 enzyme, including pH and temperature were determined. The L-phenylalanine DOT-T1E derivative Pseudomonas putida CM12-5 that overproduces L-phenylalanine was used as the host for expression of pal/psc1 genes to efficiently convert L-phenylalanine into tCA, and the aromatic carboxylic acid into styrene. The highest styrene production was achieved when the pal and psc1 genes were co-expressed as an operon in P. putida CM12-5. This construction yielded styrene production exceeding 220 mg L-1. This study serves as a successful demonstration of our strategy to tailor functional enzymes for novel host organisms, thereby broadening their metabolic capabilities. This breakthrough opens the doors to the synthesis of aromatic hydrocarbons using Pseudomonas putida as a versatile biofactory.

Competition for iron shapes metabolic antagonism between Bacillus subtilis and Pseudomonas marginalis.

Siderophores have long been implicated in sociomicrobiology as determinants of bacterial interrelations. For plant-associated genera, like Bacillus and Pseudomonas, siderophores are well known for their biocontrol functions. Here, we explored the functional role of the Bacillus subtilis siderophore bacillibactin (BB) in an antagonistic interaction with Pseudomonas marginalis. The presence of BB strongly influenced the outcome of the interaction in an iron-dependent manner. The BB producer B. subtilis restricts colony spreading of P. marginalis by repressing the transcription of histidine kinase-encoding gene gacS, thereby abolishing production of secondary metabolites such as pyoverdine and viscosin. By contrast, lack of BB restricted B. subtilis colony growth. To explore the specificity of the antagonism, we cocultured B. subtilis with a collection of fluorescent Pseudomonas spp. and found that the Bacillus-Pseudomonas interaction is conserved, expanding our understanding of the interplay between two of the most well-studied genera of soil bacteria.

Bacteria use a catabolic patchwork pathway of apparently recent origin for degradation of the synthetic buffer compound TRIS.

The synthetic buffer compound TRIS (2-amino-2-(hydroxymethyl)propane-1,3-diol) is used in countless applications, and no detailed information on its degradation has been published so far. Herein, we describe the discovery of a complete bacterial degradation pathway for TRIS. By serendipity, a Pseudomonas strain was isolated from sewage sludge that was able to grow with TRIS as only carbon and nitrogen source. Genome and transcriptome analyses revealed two adjacent gene clusters embedded in a mobile genetic element on a conjugative plasmid to be involved in TRIS degradation. Heterologous gene expression revealed cluster I to encode a TRIS uptake protein, a TRIS alcohol dehydrogenase, and a TRIS aldehyde dehydrogenase, catalyzing the oxidation of TRIS into 2-hydroxymethylserine. Gene cluster II encodes a methylserine hydroxymethyltransferase (mSHMT) and a d-serine dehydratase that plausibly catalyze the conversion of 2-hydroxymethylserine into pyruvate. Conjugational plasmid transfer into Pseudomonas putida KT2440 enabled this strain to grow with TRIS and with 2-hydromethylserine, demonstrating that the complete TRIS degradation pathway can be transmitted by horizontal gene transfer. Subsequent enrichments from wastewater purification systems led to the isolation of further TRIS-degrading bacteria from the Pseudomonas and Shinella genera carrying highly similar TRIS degradation gene clusters. Our data indicate that TRIS degradation evolved recently via gene recruitment and enzyme adaptation from multiple independent metabolic pathways, and database searches suggest that the TRIS degradation pathway is now globally distributed. Overall, our study illustrates how engineered environments can enhance the emergence of new microbial metabolic pathways in short evolutionary time scales.

Marker and readout genes for defense priming in Pseudomonas cannabina pv. alisalensis interaction aid understanding systemic immunity in Arabidopsis.

Following localized infection, the entire plant foliage becomes primed for enhanced defense. However, specific genes induced during defense priming (priming-marker genes) and those showing increased expression in defense-primed plants upon rechallenge (priming-readout genes) remain largely unknown. In our Arabidopsis thaliana study, genes AT1G76960 (function unknown), CAX3 (encoding a vacuolar Ca2+/H+ antiporter), and CRK4 (encoding a cysteine-rich receptor-like protein kinase) were strongly expressed during Pseudomonas cannabina pv. alisalensis-induced defense priming, uniquely marking the primed state for enhanced defense. Conversely, PR1 (encoding a pathogenesis-related protein), RLP23 and RLP41 (both encoding receptor-like proteins) were similarly activated in defense-primed plants before and after rechallenge, suggesting they are additional marker genes for defense priming. In contrast, CASPL4D1 (encoding Casparian strip domain-like protein 4D1), FRK1 (encoding flg22-induced receptor-like kinase), and AT3G28510 (encoding a P loop-containing nucleoside triphosphate hydrolases superfamily protein) showed minimal activation in uninfected, defense-primed, or rechallenged plants, but intensified in defense-primed plants after rechallenge. Notably, mutation in only priming-readout gene NHL25 (encoding NDR1/HIN1-like protein 25) impaired both defense priming and systemic acquired resistance, highlighting its previously undiscovered pivotal role in systemic plant immunity.

Bacillus sp. G2112 Detoxifies Phenazine-1-carboxylic Acid by N5 Glucosylation.

Microbial symbionts of plants constitute promising sources of biocontrol organisms to fight plant pathogens. Bacillus sp. G2112 and Pseudomonas sp. G124 isolated from cucumber (Cucumis sativus) leaves inhibited the plant pathogens Erwinia and Fusarium. When Bacillus sp. G2112 and Pseudomonas sp. G124 were co-cultivated, a red halo appeared around Bacillus sp. G2112 colonies. Metabolite profiling using liquid chromatography coupled to UV and mass spectrometry revealed that the antibiotic phenazine-1-carboxylic acid (PCA) released by Pseudomonas sp. G124 was transformed by Bacillus sp. G2112 to red pigments. In the presence of PCA (>40 µg/mL), Bacillus sp. G2112 could not grow. However, already-grown Bacillus sp. G2112 (OD600 > 1.0) survived PCA treatment, converting it to red pigments. These pigments were purified by reverse-phase chromatography, and identified by high-resolution mass spectrometry, NMR, and chemical degradation as unprecedented 5N-glucosylated phenazine derivatives: 7-imino-5N-(1'ß-D-glucopyranosyl)-5,7-dihydrophenazine-1-carboxylic acid and 3-imino-5N-(1'ß-D-glucopyranosyl)-3,5-dihydrophenazine-1-carboxylic acid. 3-imino-5N-(1'ß-D-glucopyranosyl)-3,5-dihydrophenazine-1-carboxylic acid did not inhibit Bacillus sp. G2112, proving that the observed modification constitutes a resistance mechanism. The coexistence of microorganisms-especially under natural/field conditions-calls for such adaptations, such as PCA inactivation, but these can weaken the potential of the producing organism against pathogens and should be considered during the development of biocontrol strategies.

Pseudomonas environmental strain produces a DegQ-derived and PDZ domain containing peptide with protease activity.

In the search of new enzymatic activities with a possible industrial application, we focused on those microorganisms and their molecular mechanisms that allow them to succeed in the environment, particularly in the proteolytic activity and its central role in the microorganisms' successful permanence. The use of highly active serine proteases for industrial applications is a modern need, especially for the formulation of detergents, protein processing, and hair removal from animal skins. This report provides the isolation and identification of a highly proteolytic fragment derived from DegQ produced by a Pseudomonas fluorescens environmental strain isolated from a frog carcass. Zymograms demonstrate that a 10 kDa protein mainly generates the total proteolytic activity of this strain, which is enhanced by the detergent SDS. Mass spectroscopy analysis revealed that the protein derived a couple of peptides, the ones showing the highest coverage belonging to DegQ. Interestingly, this small protein fragment contains a PDZ domain but no obvious residues indicating that it is a protease. Protein model analysis shows that this fragment corresponds to the main PDZ domain from DegQ, and its unique sequence and structure render a proteolytic peptide. The results presented here indicate that a novel DegQ fragment is sufficient for obtaining high protease activity highlighting that the analysis of environmental microorganisms can render new strains or enzymes with helpful biotechnological characteristics.

New insights into xenobiotic tolerance of Antarctic bacteria: transcriptomic analysis of Pseudomonas sp. TNT3 during 2,4,6-trinitrotoluene biotransformation.

The xenobiotic 2,4,6-trinitrotoluene (TNT) is a highly persistent environmental contaminant, whose biotransformation by microorganisms has attracted renewed attention. In previous research, we reported the discovery of Pseudomonas sp. TNT3, the first described Antarctic bacterium with the ability to biotransform TNT. Furthermore, through genomic analysis, we identified distinctive features in this isolate associated with the biotransformation of TNT and other xenobiotics. However, the metabolic pathways and genes active during TNT exposure in this bacterium remained unexplored. In the present transcriptomic study, we used RNA-sequencing to investigate gene expression changes in Pseudomonas sp. TNT3 exposed to 100 mg/L of TNT. The results showed differential expression of 194 genes (54 upregulated and 140 downregulated), mostly encoding hypothetical proteins. The most highly upregulated gene (> 1000-fold) encoded an azoreductase enzyme not previously described. Other significantly upregulated genes were associated with (nitro)aromatics detoxification, oxidative, thiol-specific, and nitrosative stress responses, and (nitro)aromatic xenobiotic tolerance via efflux pumps. Most of the downregulated genes were involved in the electron transport chain, pyrroloquinoline quinone (PQQ)-related alcohol oxidation, and motility. These findings highlight a complex cellular response to TNT exposure, with the azoreductase enzyme likely playing a crucial role in TNT biotransformation. Our study provides new insights into the molecular mechanisms of TNT biotransformation and aids in developing effective TNT bioremediation strategies. To the best of our knowledge, this report is the first transcriptomic response analysis of an Antarctic bacterium during TNT biotransformation.

A whole-cell hypersensitive biosensor for beta-lactams based on the AmpR-AmpC regulatory circuit from the Antarctic Pseudomonas sp. IB20.

Detecting antibiotic residues is vital to minimize their impact. Yet, existing methods are complex and costly. Biosensors offer an alternative. While many biosensors detect various antibiotics, specific ones for beta-lactams are lacking. To address this gap, a biosensor based on the AmpC beta-lactamase regulation system (ampR-ampC) from Pseudomonas sp. IB20, an Antarctic isolate, was developed in this study. The AmpR-AmpC system is well-conserved in the genus Pseudomonas and has been extensively studied for its involvement in peptidoglycan recycling and beta-lactam resistance. To create the biosensor, the ampC coding sequence was replaced with the mCherry fluorescent protein as a reporter, resulting in a transcriptional fusion. This construct was then inserted into Escherichia coli SN0301, a beta-lactam hypersensitive strain, generating a whole-cell biosensor. The biosensor demonstrated dose-dependent detection of penicillins, cephalosporins and carbapenems. However, the most interesting aspect of this work is the high sensitivity presented by the biosensor in the detection of carbapenems, as it was able to detect 8 pg/mL of meropenem and 40 pg/mL of imipenem and reach levels of 1-10 ng/mL for penicillins and cephalosporins. This makes the biosensor a powerful tool for the detection of beta-lactam antibiotics, specifically carbapenems, in different matrices.

What are the signals that control catabolite repression in Pseudomonas?

Metabolically versatile bacteria exhibit a global regulatory response known as carbon catabolite repression (CCR), which prioritizes some carbon sources over others when all are present in sufficient amounts. This optimizes growth by distributing metabolite fluxes, but can restrict yields in biotechnological applications. The molecular mechanisms and preferred substrates for CCR vary between bacterial groups. Escherichia coli prioritizes glucose whereas Pseudomonas sp. prefer certain organic acids or amino acids. A significant issue in understanding (and potentially bypassing) CCR is the lack of information about the signals that trigger this regulatory response. In E. coli, several key compounds act as flux sensors, governing the flow of metabolites through catabolic pathways and preventing imbalances. These flux sensors can also modulate the CCR response. It has been suggested that the order of substrate preference is determined by carbon uptake flux rather than substrate identity. For Pseudomonas, much less information is available, as the signals that induce CCR are poorly understood. This article briefly discusses the available evidence on the signals that trigger CCR and the questions that remain to be answered in Pseudomonas.

Genome analysis of Pseudomonas strain 4B with broad antagonistic activity against toxigenic fungi.

Pseudomonas sp. 4B isolated from the effluent pond of a bovine abattoir was investigated as antifungal against toxigenic fungi. The complete genome of Pseudomonas 4B was sequenced using the Illumina MiSeq platform. Phylogenetic analysis and genome comparisons indicated that the strain belongs to the Pseudomonas aeruginosa group. In silico investigation revealed gene clusters associated with the biosynthesis of several antifungals, including pyocyanin, rhizomide, thanamycin, and pyochelin. This bacterium was investigated through antifungal assays, showing an inhibitory effect against all toxigenic fungi tested. Bacterial cells reduced the diameter of fungal colonies, colony growth rate, and sporulation of each indicator fungi in 10-day simultaneous growing tests. The co-incubation of bacterial suspension and fungal spores in yeast extract-sucrose broth for 48 h resulted in reduced spore germination. During simultaneous growth, decreased production of aflatoxin B1 and ochratoxin A by Aspergillus flavus and Aspergillus carbonarius, respectively, was observed. Genome analysis and in vitro studies showed the ability of P. aeruginosa 4B to reduce fungal growth parameters and mycotoxin levels, indicating the potential of this bacterium to control toxigenic fungi. The broad antifungal activity of this strain may represent a sustainable alternative for the exploration and subsequent use of its possible metabolites in order to control mycotoxin-producing fungi.