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Genes Sep 2023strain U can be grown using, as sole carbon sources, the biogenic amines putrescine or cadaverine, as well as their catabolic intermediates, ɣ-aminobutyrate or...
strain U can be grown using, as sole carbon sources, the biogenic amines putrescine or cadaverine, as well as their catabolic intermediates, ɣ-aminobutyrate or δ-aminovalerate, respectively. Several paralogs for the genes that encode some of the activities involved in the catabolism of these compounds, such as a putrescine-pyruvate aminotransferase ( and genes) and a ɣ-aminobutyrate aminotransferase ( and genes) have been identified in this bacterium. When the expression pattern of these genes is analyzed by qPCR, it is drastically conditioned by supplying the carbon sources. Thus, is upregulated by putrescine, whereas seems to be exclusively induced by cadaverine. However, increases its expression in response to different polyamines or aminated catabolic derivatives from them (i.e., ɣ-aminobutyrate or δ-aminovalerate), although does not change its expression level concerning no-amine unrelated carbon sources (citrate). These results reveal differences between the mechanisms proposed for polyamine catabolism in and concerning strain U, as well as allow a deeper understanding of the enzymatic systems used by this last strain during polyamine metabolism.
Topics: Cadaverine; Putrescine; Pseudomonas putida; Polyamines; Pseudomonas aeruginosa; Escherichia coli; Aminobutyrates; Carbon; Gene Expression
PubMed: 37895246
DOI: 10.3390/genes14101897 -
International Journal of Biological... Jun 2022Polyhydroxyalkanoates (PHAs), a class of bioplastics produced by a variety of microorganisms, have become the ideal alternatives for oil-derived plastics due to their...
Polyhydroxyalkanoates (PHAs), a class of bioplastics produced by a variety of microorganisms, have become the ideal alternatives for oil-derived plastics due to their superior physicochemical and material characteristics. Pseudomonas putida KT2440 can produce medium-chain-length PHA (mcl-PHA) from various substrates. In this study, a novel strategy of the large-scale deletion of genomic islands (GIs) coupling with promoter engineering was developed in P. putida KT2440 for constructing the minimal genome cell factories (MGF) capable of efficiently producing mcl-PHA. Firstly, P. putida KTU-U13, a 13 GIs- and upp-deleted mutant derived from the parental strain P. putida KT2440, was used as a starting strain for further deletion of GIs to generate a series of genome-reduced strains. Subsequently, the two minimal genome strains KTU-U24 and KTU-U27, which had a 7.19% and 8.35% reduction relative to the genome size of KT2440 and were advantageous over the strain KTU (KT2440∆upp) and KTU-U13 in several physiological traits such as the maximum specific growth rate, plasmid transformation efficiency, heterologous protein expression capacity and PHA production capacity, were selected as the chassis cells for PHA metabolic engineering. To prevent the formation of the by-product gluconic acid, the glucose dehydrogenase gene was deleted in KTU-U24 and KTU-U27, resulting in KTU-U24∆gcd and KTU-U27∆gcd. To enhance the transcriptional level of PHA synthase genes (phaC) and the supply of the precursor acetyl-CoA, a strong endogenous promoter P46 was inserted into upstream of the phaC operon and pyruvate dehydrogenase gene in the genome of KTU-U24∆gcd and KTU-U27∆gcd, to generate KTU-U24∆gcd-P46CA and KTU-U27∆gcd-P46CA, with the PHA yield of 50.5 wt% and 53.8 wt% (weight percent of PHA in cell dry weight). Finally, KTU-U27∆gcd-P46CA, the most minimal KT2440 chassis currently available, was able to accumulate the PHA to 55.82 wt% in a 5-l fermentor, which is the highest PHA yield obtained with P. putida KT2440 so far. This study suggests that genome streamlining in combination with promoter engineering may be a feasible strategy for the development of the MGF for the efficient production of high value products. Moreover, further streamlining of the P. putida KT2440 genome has great potential to create the optimal chassis for synthetic biology applications.
Topics: Metabolic Engineering; Polyhydroxyalkanoates; Promoter Regions, Genetic; Pseudomonas putida; Synthetic Biology
PubMed: 35395277
DOI: 10.1016/j.ijbiomac.2022.04.004 -
Applied and Environmental Microbiology Apr 2022The second messenger cyclic di-GMP (c-di-GMP) is a key molecule that controls different physiological and behavioral processes in many bacteria, including...
The second messenger cyclic di-GMP (c-di-GMP) is a key molecule that controls different physiological and behavioral processes in many bacteria, including motile-to-sessile lifestyle transitions. Although the external stimuli that modulate cellular c-di-GMP contents are not fully characterized, there is growing evidence that certain amino acids act as environmental cues for c-di-GMP turnover. In the plant-beneficial bacterium Pseudomonas putida KT2440, both arginine biosynthesis and uptake influence second messenger contents and the associated phenotypes. To further understand this connection, we have analyzed the role of ArgR, which in different bacteria is the master transcriptional regulator of arginine metabolism but had not been characterized in P. putida. The results show that ArgR controls arginine biosynthesis and transport, and an -null mutant grows poorly with arginine as the sole carbon or nitrogen source and also displays increased biofilm formation and reduced surface motility. Modulation of c-di-GMP levels by exogenous arginine requires ArgR. The expression of certain biofilm matrix components, namely, the adhesin LapF and the exopolysaccharide Pea, as well as the diguanylate cyclase CfcR is influenced by ArgR, likely through the alternative sigma factor RpoS. Our data indicate the existence of a regulatory feedback loop between ArgR and c-di-GMP mediated by FleQ. Identifying the molecular mechanisms by which metabolic and environmental signals influence the turnover of the second messenger c-di-GMP is key to understanding the regulation of bacterial lifestyles. The results presented here point at the transcriptional regulator ArgR as a central node linking arginine metabolism and c-di-GMP signaling and indicate the existence of a complex balancing mechanism that connects cellular arginine contents and second messenger levels, ultimately controlling the lifestyles of Pseudomonas putida.
Topics: Arginine; Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Pseudomonas putida
PubMed: 35254100
DOI: 10.1128/aem.00064-22 -
Microbial Biotechnology Oct 2022The extracellular 373-kDa PehA heme peroxidase of Pseudomonas putida KT2440 has two enzymatic domains which depend on heme cofactor for their peroxidase activity. A null...
The extracellular 373-kDa PehA heme peroxidase of Pseudomonas putida KT2440 has two enzymatic domains which depend on heme cofactor for their peroxidase activity. A null pehA mutant was generated to examine the impact of PehA in rhizosphere colonization competence and the induction of plant systemic resistance (ISR). This mutant was not markedly hampered in colonization efficiency. However, increase in pehA dosage enhanced colonization fitness about 30 fold in the root and 900 fold in the root apex. In vitro assays with purified His-tagged enzymatic domains of PehA indicated that heme-dependent peroxidase activity was required for the enhancement of root tip colonization. Evaluation of live/dead cells confirmed that overexpression of pehA had a positive effect on bacterial cell viability. Following root colonization of rice plants by KT2440 strain, the incidence of rice blast caused by Magnaporthe oryzae was reduced by 65% and the severity of this disease was also diminished in comparison to non-treated plants. An increase in the pehA dosage was also beneficial for the control of rice blast as compared with gene inactivation. The results suggest that PehA helps P. putida to cope with the plant-imposed oxidative stress leading to enhanced colonization ability and concomitant ISR-elicitation.
Topics: Antioxidants; Heme; Peroxidases; Plant Diseases; Plant Roots; Pseudomonas putida
PubMed: 35986900
DOI: 10.1111/1751-7915.14123 -
Biotechnology Letters Feb 2022Functional characterization of metagenomic DNA often involves expressing heterologous DNA in genetically tractable microorganisms such as Escherichia coli. Functional...
Functional characterization of metagenomic DNA often involves expressing heterologous DNA in genetically tractable microorganisms such as Escherichia coli. Functional expression of heterologous genes can suffer from limitations due to the lack of recognition of foreign promoters or presence of intrinsic terminators on foreign DNA between a vector-based promoter and the transcription start site. Anti-terminator proteins are a possible solution to overcome this limitation. When bacteriophage lambda infects E. coli, it relies on the host transcription machinery to transcribe and express phage DNA. Lambda anti-terminator protein Q (λQ) regulates the expression of late-genes of phage lambda. E. coli RNA polymerase recognizes the P' promoter on the lambda genome and forms a complex with λQ, to overcome the terminator t'. Here we show the use of λQ to efficiently transcribe a capsular polysaccharide cluster, cps3, from Lactobacillus plantarum containing intrinsic terminators in Escherichia coli. In addition, we expand the use of anti-terminator λQ in Pseudomonas putida. The results show ~ fivefold higher expression of a fluorescent reporter located ~ 12.5kbp downstream from the promoter, when the transcription is driven by P' promoter in presence of λQ compared to a lac promoter. These results suggest that λQ could be used in metabolic engineering to enhance expression of heterologous DNA.
Topics: Bacterial Proteins; Bacteriophage lambda; DNA-Directed RNA Polymerases; Escherichia coli; Promoter Regions, Genetic; Pseudomonas putida; Transcription, Genetic
PubMed: 34792701
DOI: 10.1007/s10529-021-03206-x -
Environmental Microbiology Feb 2023The Pseudomonas putida group in the Gammaproteobacteria has been intensively studied for bioremediation and plant growth promotion. Members of this group have recently...
The Pseudomonas putida group in the Gammaproteobacteria has been intensively studied for bioremediation and plant growth promotion. Members of this group have recently emerged as promising hosts to convert intermediates derived from plant biomass to biofuels and biochemicals. However, most strains of P. putida cannot metabolize pentose sugars derived from hemicellulose. Here, we describe three isolates that provide a broader view of the pentose sugar catabolism in the P. putida group. One of these isolates clusters with the well-characterized P. alloputida KT2440 (Strain BP6); the second isolate clustered with plant growth-promoting strain P. putida W619 (Strain M2), while the third isolate represents a new species in the group (Strain BP8). Each of these isolates possessed homologous genes for oxidative xylose catabolism (xylDXA) and a potential xylonate transporter. Strain M2 grew on arabinose and had genes for oxidative arabinose catabolism (araDXA). A CRISPR interference (CRISPRi) system was developed for strain M2 and identified conditionally essential genes for xylose growth. A glucose dehydrogenase was found to be responsible for initial oxidation of xylose and arabinose in strain M2. These isolates have illuminated inherent diversity in pentose catabolism in the P. putida group and may provide alternative hosts for biomass conversion.
Topics: Pentoses; Xylose; Arabinose; Pseudomonas putida; Oxidative Stress
PubMed: 36465038
DOI: 10.1111/1462-2920.16296 -
Medium-chain alkane biodegradation and its link to some unifying attributes of alkB genes diversity.The Science of the Total Environment Jun 2023Hydrocarbon footprints in the environment, via biosynthesis, natural seepage, anthropogenic activities and accidents, affect the ecosystem and induce a shift in the... (Review)
Review
Hydrocarbon footprints in the environment, via biosynthesis, natural seepage, anthropogenic activities and accidents, affect the ecosystem and induce a shift in the healthy biogeochemical equilibrium that drives needed ecological services. In addition, these imbalances cause human diseases and reduce animal and microorganism diversity. Microbial bioremediation, which capitalizes on functional genes, is a sustainable mitigation option for cleaning hydrocarbon-impacted environments. This review focuses on the bacterial alkB functional gene, which codes for a non-heme di‑iron monooxygenase (AlkB) with a di‑iron active site that catalyzes C-C medium-chain alkane metabolism. These enzymes are ubiquitous and share common attributes such as being controlled by global transcriptional regulators, being a component of most super hydrocarbon degraders, and their distributions linked to horizontal gene transfer (HGT) events. The phylogenetic approach used in the HGT detection suggests that AlkB tree topology clusters bacteria functionally and that a preferential gradient dictates gene distribution. The alkB gene also acts as a biomarker for bioremediation, although it is found in pristine environments and absent in some hydrocarbon degraders. For instance, a quantitative molecular method has failed to link alkB copy number to contamination concentration levels. This limitation may be due to AlkB homologues, which have other functions besides n-alkane assimilation. Thus, this review, which focuses on Pseudomonas putida GPo1 alkB, shows that AlkB proteins are diverse but have some unifying trends around hydrocarbon-degrading bacteria; it is erroneous to rely on alkB detection alone as a monitoring parameter for hydrocarbon degradation, alkB gene distribution are preferentially distributed among bacteria, and the plausible explanation for AlkB affiliation to broad-spectrum metabolism of hydrocarbons in super-degraders hitherto reported. Overall, this review provides a broad perspective of the ecology of alkB-carrying bacteria and their directed biodegradation pathways.
Topics: Animals; Humans; Alkanes; Biodegradation, Environmental; Ecosystem; Hydrocarbons; Iron; Phylogeny; Pseudomonas putida; Genes, Bacterial
PubMed: 36948313
DOI: 10.1016/j.scitotenv.2023.162951 -
Journal of Applied Microbiology Jul 2021To isolate, identify and characterize phenolic acid-degrading bacteria and reduce plant growth inhibition caused by phenolic acids.
AIMS
To isolate, identify and characterize phenolic acid-degrading bacteria and reduce plant growth inhibition caused by phenolic acids.
METHODS AND RESULTS
A total of 11 bacterial isolates with high phthalic acid (PA)-degrading ability were obtained using mineral salt medium (MSM) medium containing PA as sole carbon source. These isolates were identified as Arthrobacter globiformis, Pseudomonas putida and Pseudomonas hunanensis by sequence analyses of the 16S rRNA gene. Among them, five Pseudomonas strains could also effectively degrade ferulic acid (FA), p-hydroxybenzoic acid (PHBA) and syringic acid (SA) in MSM solution. P. putida strain 7 and P. hunanensis strain 10 showed highly efficient degradation of PA, SA, FA and PHBA, and could reduce their inhibition of lily, watermelon, poplar and strawberry seedling growth in soils respectively. These two strains could promote plant growth in soil with phenolic acids.
CONCLUSIONS
In this study, bacterial strains with highly efficient phenolic acid-degrading abilities could not only effectively reduce the autotoxicity of phenolic acids on plants but also were able to promote plant growth in soil with phenolic acids.
SIGNIFICANCE AND IMPACT OF THE STUDY
In this study, Pseudomonas can promote plant growth while degrading phenolic acids. Our results provide new choices for the biological removal of autotoxins.
Topics: Arthrobacter; Biodegradation, Environmental; Coumaric Acids; Gallic Acid; Hydroxybenzoates; Parabens; Phylogeny; Plant Development; Pseudomonas; Pseudomonas putida; RNA, Ribosomal, 16S; Rhizosphere; Seedlings; Soil Microbiology
PubMed: 33270328
DOI: 10.1111/jam.14956 -
Environmental Pollution (Barking, Essex... Feb 2021Antibiotics are frequently used for clinical treatment and by the farming industry, and most of these are eventually released into the surrounding environment. The...
Antibiotics are frequently used for clinical treatment and by the farming industry, and most of these are eventually released into the surrounding environment. The impact of these antibiotic pollutants on environmental microorganisms is a concern. The present study showed that after Pseudomonas putida entered the logarithmic growth phase, tetracycline strongly stimulated its biofilm formation in a dose-dependent manner. This was supported by the increased expression of the key adhesin gene lapA in response to tetracycline treatment. Tetracycline treatment also changed the expression levels of the exopolysaccharide gene clusters alg, bcs and pea and the adhesin gene lapF. However, these genes did not participate in the tetracycline-induced biofilm formation. When a biofilm had been established, the P. putida population became more tolerant to tetracycline. Confocal laser scanning microscopic images showed that the interior of the biofilm provided favorable conditions that protected bacterial cells from tetracycline. Besides, biofilm formation of P. putida was also promoted by several other antibiotics, including oxytetracycline, fluoroquinolones, rifampicin, and imipenem, but not aminoglycosides. Susceptibility tests suggested that biofilm conferred a higher tolerance on P. putida cells to specific antibiotics (e.g., tetracyclines and fluoroquinolones). These antibiotics exerted a stronger inducing effect on biofilm formation. Together, our results indicate that P. putida actively forms robust biofilms in response to antibiotic stress, and the biofilms improve the survival of bacterial population under such stress.
Topics: Adhesins, Bacterial; Anti-Bacterial Agents; Biofilms; Pseudomonas putida
PubMed: 33359874
DOI: 10.1016/j.envpol.2020.116261 -
PloS One 2019Pseudomonas putida is one of 13 major groups of Pseudomonas spp. and contains numerous species occupying diverse niches and performing many functions such as plant...
Pseudomonas putida is one of 13 major groups of Pseudomonas spp. and contains numerous species occupying diverse niches and performing many functions such as plant growth promotion and bioremediation. Here we compared a set of 19 P. putida isolates obtained from sugarcane rhizosphere or bulk soil using a population genomics approach aiming to assess genomic and metabolic differences between populations from these habitats. Phylogenomics placed rhizosphere versus bulk soil strains in separate clades clustering with different type strains of the P. putida group. Multivariate analyses indicated that the rhizosphere and bulk soil isolates form distinct populations. Comparative genomics identified several genetic functions (GO-terms) significantly different between populations, including some exclusively present in the rhizosphere or bulk soil strains, such as D-galactonic acid catabolism and cellulose biosynthesis, respectively. The metabolic profiles of rhizosphere and bulk soil populations analyzed by Biolog Ecoplates also differ significantly, most notably by the higher oxidation of D-galactonic/D-galacturonic acid by the rhizosphere population. Accordingly, D-galactonate catabolism operon (dgo) was present in all rhizosphere isolates and absent in the bulk soil population. This study showed that sugarcane rhizosphere and bulk soil harbor different populations of P. putida and identified genes and functions potentially associated with their soil niches.
Topics: Antibiosis; Genetics, Population; Genome, Bacterial; Genomics; Metabolomics; Phylogeny; Pseudomonas putida; Rhizosphere; Saccharum; Soil Microbiology
PubMed: 31581220
DOI: 10.1371/journal.pone.0223269