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Journal of Bacteriology Jan 2022The transcriptomes of Pseudomonas aeruginosa clone C isolates NN2 and SG17M during the mid-exponential and early stationary phases of planktonic growth were evaluated by...
The transcriptomes of Pseudomonas aeruginosa clone C isolates NN2 and SG17M during the mid-exponential and early stationary phases of planktonic growth were evaluated by direct RNA sequencing on the nanopore platform and compared with established short-read cDNA sequencing on the Illumina platform. Fifty to ninety percent of the sense RNAs turned out to be rRNA molecules, followed by similar proportions of mRNA transcripts and noncoding RNAs. The two platforms detected similar proportions of uncharged tRNAs and 29 yet-undescribed antisense tRNAs. For example, the rarest arginine codon was paired with the most abundant tRNA, and the tRNA gene is missing for the most frequent arginine codon. More than 90% of the antisense RNA molecules were complementary to a coding sequence. The antisense RNAs were evenly distributed in the genomes. Direct RNA sequencing identified more than 4,000 distinct nonoverlapping antisense RNAs during exponential and stationary growth. Besides highly expressed small antisense RNAs less than 200 bases in size, a population of longer antisense RNAs was sequenced that covered a broad range (a few hundred to thousands of bases) and could be complementary to a contig of several genes. In summary, direct RNA sequencing identified yet-undescribed RNA molecules and an unexpected composition of the pools of tRNAs and sense and antisense RNAs. Genome-wide gene expression of bacteria is commonly studied by high-throughput sequencing of size-selected cDNA fragment libraries of reverse-transcribed RNA preparations. However, the depletion of rRNAs, enzymatic reverse transcription, and the fragmentation, size selection, and amplification during library preparation lead to inevitable losses of information about the initial composition of the RNA pool. We demonstrate that direct RNA sequencing on the Nanopore platform can overcome these limitations. Nanopore sequencing of total RNA yielded novel insights into the Pseudomonas aeruginosa transcriptome that-if replicated in other species-will change our view of the bacterial RNA world. The discovery of sense-antisense pairs of transfer-messenger RNA (tmRNA), tRNAs, and mRNAs indicates a further and unknown level of gene regulation in bacteria.
Topics: Gene Expression Regulation, Bacterial; Genome, Bacterial; Genome-Wide Association Study; Nanopore Sequencing; Pseudomonas aeruginosa; RNA, Bacterial; Transcriptome
PubMed: 34780302
DOI: 10.1128/JB.00418-21 -
MBio Dec 2021Perfluorinated carbon atoms in a diether linkage are common in commercial anesthetics, drugs, fungicides, and insecticides. An important chemical group comprising...
Perfluorinated carbon atoms in a diether linkage are common in commercial anesthetics, drugs, fungicides, and insecticides. An important chemical group comprising perfluorodiethers is the 2,2-fluoro-1,3-benzodioxole (DFBD) moiety. The fluorine atoms stabilize the molecule by mitigating against metabolism by humans and microbes, as used in drugs and pesticides, respectively. Pseudomonas putida F1 catalyzed defluorination of DFBD at an initial rate of 2,100 nmol/h per mg cellular protein. This is orders of magnitude higher than previously reported microbial defluorination rates with multiply fluorinated carbon atoms. Defluorination rates declined after several hours, and the medium darkened. Significant defluorination activity was observed with cells grown on toluene but not l-arginine. Defluorination required only toluene dioxygenase. Pseudomonas and recombinant Escherichia coli cells expressing toluene dioxygenase oxidized DFBD to DFBD-4,5-dihydrodiol. The dihydrodiol could be oxidized to 4,5-dihydroxy-DFBD via the dihydrodiol dehydrogenase from P. putida F1. The dihydrodiol dehydrated with acid to yield a mixture of 4-hydroxy-DFBD and 5-hydroxy-DFBD. All those metabolites retained the difluoromethylene group; no fluoride or dark color was observed. The major route of DFBD-4,5-dihydrodiol decomposition produced fluoride and 1,2,3-trihydroxybenzene, or pyrogallol, and that was shown to be the source of the dark colors in the medium. A mechanism for DFBD-4,5-dihydrodiol transformation to two fluoride ions and pyrogallol is proposed. The Pseudomonas genome database and other databases revealed hundreds of bacteria with enzymes sharing high amino acid sequence identity to toluene dioxygenase from P. putida F1, suggesting the mechanism revealed here may apply to the defluorination of DFBD-containing compounds in the environment. There are more than 9,000 polyfluorinated compounds developed for commercial use, some negatively impacting human health, and they are generally considered to be resistant to biodegradation. Only a limited number of studies have identified microbes with enzymes sufficiently reactive to defluorinate difluoromethylene carbon groups. The present study examined one important group of commercial fluorinated chemicals and showed its rapid defluorination by a bacterium and its key enzyme, a Rieske dioxygenase. Rieske dioxygenases are common in environmental bacteria, and those closely resembling toluene dioxygenase from Pseudomonas putida F1 are candidates for biodegradative defluorination of the common 2,2-fluoro-1,3-benzodioxole (DFBD) moiety.
Topics: Bacterial Proteins; Biodegradation, Environmental; Dioxoles; Halogenation; Oxygenases; Pseudomonas putida
PubMed: 34781746
DOI: 10.1128/mBio.03001-21 -
Scientific Reports Jun 2024A novel nano bio-fertilizer encapsulation method was developed to crosslink chitosan and alginate with humic acid. These nanocapsules, referred to as (Ch./Alg.HA.NPK) or...
A novel nano bio-fertilizer encapsulation method was developed to crosslink chitosan and alginate with humic acid. These nanocapsules, referred to as (Ch./Alg.HA.NPK) or (Ch./Alg.HA.NPK.PGPRs), were loaded with nanoscale essential agro-nutrients (NPK) and beneficial microorganisms Pseudomonas Fluorescence abbreviated as (P.Fluorescence). Structural and morphological analyses were conducted using FourierTransform Infrared, Thermogravimetric Analysis, Scanning Electron Microscopy, Malvern Zeta NanoSizer, and Zeta potential. Encapsulation efficiency and water retention were also determined compared to control non-crosslinked nanocapsules. The sustained cumulative release of NPK over 30 days was also investigated to 33.2%, 47.8%, and 68.3%, alternatively. The release mechanism, also assessed through the kinetic module of the Korsemeyer- Peppas Mathematical model, demonstrated superior performance compared to non-crosslinked nanocapsules (chitosan/alginate). These results show the potential of the synthesized nanocapsules for environmentally conscious controlled release of NPK and PGPRs, thereby mitigating environmental impact, enhancing plant growth, and reducing reliance on conventional agrochemical fertilizers.
Topics: Fertilizers; Chitosan; Agriculture; Alginates; Nanocapsules; Humic Substances; Pseudomonas
PubMed: 38871758
DOI: 10.1038/s41598-024-62973-5 -
Microbial Biotechnology Jan 2022To meet the needs of synthetic biologists, DNA assembly methods have transformed from simple 'cut-and-paste' procedures to highly advanced, standardised assembly...
To meet the needs of synthetic biologists, DNA assembly methods have transformed from simple 'cut-and-paste' procedures to highly advanced, standardised assembly techniques. Implementing these standardised DNA assembly methods in biotechnological research conducted in non-model hosts, including Pseudomonas putida and Pseudomonas aeruginosa, could greatly benefit reproducibility and predictability of experimental results. SEVAtile is a Type IIs-based assembly approach, which enables the rapid and standardised assembly of genetic parts - or tiles - to create genetic circuits in the established SEVA-vector backbone. Contrary to existing DNA assembly methods, SEVAtile is an easy and straightforward method, which is compatible with any vector, both SEVA- and non-SEVA. To prove the efficiency of the SEVAtile method, a three-vector system was successfully generated to independently co-express three different proteins in P. putida and P. aeruginosa. More specifically, one of the vectors, pBGDes, enables genomic integration of assembled circuits in the Tn7 landing site, while self-replicatory vectors pSTDesX and pSTDesR enable inducible expression from the XylS/Pm and RhaRS/PrhaB expression systems, respectively. Together, we hope these vector systems will support research in both the microbial SynBio and Pseudomonas field.
Topics: DNA; Genetic Vectors; Plasmids; Pseudomonas; Pseudomonas putida; Reproducibility of Results
PubMed: 34651450
DOI: 10.1111/1751-7915.13922 -
BMC Microbiology Apr 2021Faba bean (Vicia faba L.) cultivation is highly challenged by faba bean black root rot disease (Fusarium solani) in high lands of Ethiopia. To ensure sustainable...
In-vitro compatibility assay of indigenous Trichoderma and Pseudomonas species and their antagonistic activities against black root rot disease (Fusarium solani) of faba bean (Vicia faba L.).
BACKGROUND
Faba bean (Vicia faba L.) cultivation is highly challenged by faba bean black root rot disease (Fusarium solani) in high lands of Ethiopia. To ensure sustainable production of faba beans, searching for eco-friendly disease management options is necessary to curb the progress of the disease timely. The indigenous biocontrol agents that suit local environments may effectively strive with in-situ microorganisms and suppress local pathogen strains. This study aimed to screen antagonistic indigenous compatible Trichoderma and Pseudomonas strains against Fusarium solani. In the pathogenicity test, soil-filled pots were arranged in complete random block design and sown with health faba bean seeds. The effect of some fungicides was evaluated against Fusarium by food poisoning methods to compare with the biocontrol agents. The antagonistic efficacy of biocontrol agents and their compatibility was investigated on Potato dextrose agar medium.
RESULTS
Fusarium solani AAUF51 strain caused an intense root rotting in faba bean plant. The effect of Mancozeb 80% WP at 300 ppm was comparable with Trichoderma and Pseudomonas strains against Fusarium. The mycelial growth of test the pathogen was significantly (P ≤ 0.05) reduced to 86.67 and 85.19% by Trichoderma harzianum AAUW1 and Trichoderma viridae AAUC22 strains in dual culture, respectively. The volatile metabolites of Pseudomonas aeruginosa AAUS31 (77.78%) found the most efficient in reducing mycelial growth of Fusarium followed by Pseudomonas fluorescens AAUPF62 (71.11%) strains. The cell-free culture filtrates of Pseudomonas fluorescens AAUPF62 and Pseudomonas aeruginosa AAUS31 were more efficient than the Trichoderma strain in reducing the growth of Fusarium isolates. There was no zone of inhibition recorded between Trichoderma harzianum AAUW1, Trichoderma viridae AAUC22, Pseudomonas aeruginosa AAUS31, and Pseudomonas fluorescens AAUPF62 strains, hence they were mutually compatible.
CONCLUSIONS
The compatible Trichoderma and Pseudomonas strains showed antagonistic potentiality that could be explored for faba bean protection against black root rot disease and might have a future dual application as biocontrol agents.
Topics: Microbial Interactions; Pest Control, Biological; Plant Diseases; Plant Roots; Pseudomonas; Soil Microbiology; Trichoderma; Vicia faba
PubMed: 33865331
DOI: 10.1186/s12866-021-02181-7 -
Analytical Chemistry Aug 2023Small proteins of around 50 aa in length have been largely overlooked in genetic and biochemical assays due to the inherent challenges with detecting and characterizing...
Small proteins of around 50 aa in length have been largely overlooked in genetic and biochemical assays due to the inherent challenges with detecting and characterizing them. Recent discoveries of their critical roles in many biological processes have led to an increased recognition of the importance of small proteins for basic research and as potential new drug targets. One example is CcoM, a 36 aa subunit of the -type oxidase that plays an essential role in adaptation to oxygen-limited conditions in , a model for the clinically relevant, opportunistic pathogen . However, as no comprehensive data were available in , we devised an integrated, generic approach to study small proteins more systematically. Using the first complete genome as basis, we conducted bottom-up proteomics analyses and established a digest-free, direct-sequencing proteomics approach to study cells grown under aerobic and oxygen-limiting conditions. Finally, we also applied a proteogenomics pipeline to identify missed protein-coding genes. Overall, we identified 2921 known and 29 novel proteins, many of which were differentially regulated. Among 176 small proteins 16 were novel. Direct sequencing, featuring a specialized precursor acquisition scheme, exhibited advantages in the detection of small proteins with higher (up to 100%) sequence coverage and more spectral counts, including sequences with high proline content. Three novel small proteins, uniquely identified by direct sequencing and not conserved beyond , were predicted to form an operon with a conserved protein and may represent genes. These data demonstrate the power of this combined approach to study small proteins in and show its potential for other prokaryotes.
Topics: Pseudomonas stutzeri; Proteomics; Proteogenomics; Pseudomonas aeruginosa; Oxygen
PubMed: 37535005
DOI: 10.1021/acs.analchem.3c00676 -
Genes Jun 2023is a bacterial genus with some saprophytic species from land and others associated with opportunistic infections in humans and animals. Factors such as pathogenicity or...
is a bacterial genus with some saprophytic species from land and others associated with opportunistic infections in humans and animals. Factors such as pathogenicity or metabolic aspects have been related to CRISPR-Cas, and in silico studies into it have focused more on the clinical and non-environmental setting. This work aimed to perform an in silico analysis of the CRISPR-Cas systems present in genomes. It analyzed 275 complete genomic sequences of taken from the NCBI database. CRISPR loci were obtained from CRISPRdb. The genes associated with CRISPR () and CAS proteins, and the origin and diversity of spacer sequences, were identified and compared by BLAST. The presence of self-targeting sequences, PAMs, and the conservation of DRs were visualized using WebLogo 3.6. The CRISPR-like RNA secondary structure prediction was analyzed using RNAFold and MFold. CRISPR structures were identified in 19.6% of species. In all, 113 typical CRISPR arrays with 18 putative were found, as were 2050 spacers, of which 52% showed homology to bacteriophages, 26% to chromosomes, and 22% to plasmids. No potential self-targeting was detected within the CRISPR array. All the found DRs can form thermodynamically stable secondary RNA structures. The comparison of the CRISPR/Cas system can help understand the environmental adaptability of each evolutionary lineage of clinically and environmentally relevant species, providing data support for bacterial typing, traceability, analysis, and exploration of unconventional CRISPR.
Topics: Humans; Genome, Bacterial; CRISPR-Cas Systems; Pseudomonas; Plasmids; RNA
PubMed: 37510242
DOI: 10.3390/genes14071337 -
BMC Microbiology Jan 2022Peanut stem rot is a serious plant disease that causes great economic losses. At present, there are no effective measures to prevent or control the occurrence of this...
BACKGROUND
Peanut stem rot is a serious plant disease that causes great economic losses. At present, there are no effective measures to prevent or control the occurrence of this plant disease. Biological control is one of the most promising plant disease control measures. In this study, Pseudomonas chlororaphis subsp. aurantiaca strain zm-1, a bacterial strain with potential biocontrol properties isolated by our team from the rhizosphere soil of Anemarrhena asphodeloides, was studied to control this plant disease.
METHODS
We prepared extracts of Pseudomonas chloroaphis zm-1 extracellular antibacterial compounds (PECEs), determined their antifungal activities by confrontation assay, and identified their components by UPLC-MS/MS. The gene knockout strains were constructed by homologous recombination, and the biocontrol efficacy of P. chlororaphis zm-1 and its mutant strains were evaluated by pot experiments under greenhouse conditions and plot experiments, respectively.
RESULTS
P. chlororaphis zm-1 could produce extracellular antifungal substances and inhibit the growth of Sclerotium rolfsii, the main pathogenic fungus causing peanut stem rot. The components of PECEs identified by UPLC-MS/MS showed that three kinds of phenazine compounds, i.e., 1-hydroxyphenazine, phenazine-1-carboxylic acid (PCA), and the core phenazine, were the principal components. In particular, 1-hydroxyphenazine produced by P. chlororaphis zm-1 showed antifungal activities against S. rolfsii, but 2-hydroxyphenazine did not. This is quite different with the previously reported. The extracellular compounds of two mutant strains, ΔphzH and ΔphzE, was analysed and showed that ΔphzE did not produce any phenazine compounds, and ΔphzH no longer produced 1-hydroxyphenazine but could still produce PCA and phenazine. Furthermore, the antagonistic ability of ΔphzH declined, and that of ΔphzE was almost completely abolished. According to the results of pot experiments under greenhouse conditions, the biocontrol efficacy of ΔphzH dramatically declined to 47.21% compared with that of wild-type P. chlororaphis zm-1 (75.63%). Moreover, ΔphzE almost completely lost its ability to inhibit S. rolfsii (its biocontrol efficacy was reduced to 6.19%). The results of the larger plot experiments were also consistent with these results.
CONCLUSIONS
P. chlororaphis zm-1 has the potential to prevent and control peanut stem rot disease. Phenazines produced and secreted by P. chlororaphis zm-1 play a key role in the control of peanut stem rot caused by S. rolfsii. These findings provide a new idea for the effective prevention and treatment of peanut stem rot.
Topics: Antibiosis; Antifungal Agents; Arachis; Bacterial Proteins; Basidiomycota; Biological Control Agents; Mutation; Phenazines; Plant Diseases; Pseudomonas
PubMed: 34986788
DOI: 10.1186/s12866-021-02420-x -
International Journal of Molecular... Feb 2023Although many bacterial lipases and PHA depolymerases have been identified, cloned, and characterized, there is very little information on the potential application of...
Although many bacterial lipases and PHA depolymerases have been identified, cloned, and characterized, there is very little information on the potential application of lipases and PHA depolymerases, especially intracellular enzymes, for the degradation of polyester polymers/plastics. We identified genes encoding an intracellular lipase (LIP3), an extracellular lipase (LIP4), and an intracellular PHA depolymerase (PhaZ) in the genome of the bacterium PA23. We cloned these genes into and then expressed, purified, and characterized the biochemistry and substrate preferences of the enzymes they encode. Our data suggest that the LIP3, LIP4, and PhaZ enzymes differ significantly in their biochemical and biophysical properties, structural-folding characteristics, and the absence or presence of a lid domain. Despite their different properties, the enzymes exhibited broad substrate specificity and were able to hydrolyze both short- and medium-chain length polyhydroxyalkanoates (PHAs), para-nitrophenyl (pNP) alkanoates, and polylactic acid (PLA). Gel Permeation Chromatography (GPC) analyses of the polymers treated with LIP3, LIP4, and PhaZ revealed significant degradation of both the biodegradable as well as the synthetic polymers poly(ε-caprolactone) (PCL) and polyethylene succinate (PES).
Topics: Pseudomonas; Carboxylic Ester Hydrolases; Lipase; Polyesters; Polyhydroxyalkanoates; Pseudomonas chlororaphis; Substrate Specificity
PubMed: 36901931
DOI: 10.3390/ijms24054501 -
Nucleic Acids Research Dec 2023The widespread Pseudomonas genus comprises a collection of related species with remarkable abilities to degrade plastics and polluted wastes and to produce a broad...
The widespread Pseudomonas genus comprises a collection of related species with remarkable abilities to degrade plastics and polluted wastes and to produce a broad set of valuable compounds, ranging from bulk chemicals to pharmaceuticals. Pseudomonas possess characteristics of tolerance and stress resistance making them valuable hosts for industrial and environmental biotechnology. However, efficient and high-throughput genetic engineering tools have limited metabolic engineering efforts and applications. To improve their genome editing capabilities, we first employed a computational biology workflow to generate a genus-specific library of potential single-stranded DNA-annealing proteins (SSAPs). Assessment of the library was performed in different Pseudomonas using a high-throughput pooled recombinase screen followed by Oxford Nanopore NGS analysis. Among different active variants with variable levels of allelic replacement frequency (ARF), efficient SSAPs were found and characterized for mediating recombineering in the four tested species. New variants yielded higher ARFs than existing ones in Pseudomonas putida and Pseudomonas aeruginosa, and expanded the field of recombineering in Pseudomonas taiwanensisand Pseudomonas fluorescens. These findings will enhance the mutagenesis capabilities of these members of the Pseudomonas genus, increasing the possibilities for biotransformation and enhancing their potential for synthetic biology applications. .
Topics: DNA, Single-Stranded; Gene Editing; Metabolic Engineering; Pseudomonas; Pseudomonas putida
PubMed: 37941137
DOI: 10.1093/nar/gkad1024