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Microbial Biotechnology Jan 2024Rhamnolipids (RL) are biosurfactants naturally produced by the opportunistic pathogen Pseudomonas aeruginosa. Currently, RL are commercialized for various applications...
Rhamnolipids (RL) are biosurfactants naturally produced by the opportunistic pathogen Pseudomonas aeruginosa. Currently, RL are commercialized for various applications and produced by Pseudomonas putida due to the health risks associated with their large-scale production by P. aeruginosa. In this work, we show that RL containing one or two rhamnose moieties (mono-RL or di-RL, respectively) can be produced by the innocuous soil-bacterium Pseudomonas chlororaphis subsp chlororaphis ATCC 9446 at titres up to 66 mg/L (about 86% of the production of P. aeruginosa PAO1 in the same culture conditions). The production of RL depends on the expression of P. aeruginosa PAO1 genes encoding the enzymes RhlA, RhlB and RhlC. These genes were introduced in a plasmid, together with a transcriptional regulator (rhlR) forming part of the same operon, with and without RhlC. We show that the activation of rhlAB by RhlR depends on its interaction with P. chlororaphis endogenous acyl-homoserine lactones, which are synthetized by either PhzI or CsaI autoinducer synthases (producing 3-hydroxy-hexanoyl homoserine lactone, 3OH-C6-HSL, or 3-oxo-hexanoyl homoserine lactone, 3O-C6-HSL, respectively). P. chlororaphis transcriptional regulator couple with 3OH-C6-HSL is the primary activator of gene expression for phenazine-1-carboxylic acid (PCA) and phenazine-1-carboxamide (PCN) production in this soil bacterium. We show that RhlR coupled with 3OH-C6-HSL or 3O-C6-HSL promotes RL production and increases the production of PCA in P. chlororaphis. However, PhzR/3OH-C6-HSL or CsaR/3O-C6-HSL cannot activate the expression of the rhlAB operon to produce mono-RL. These results reveal a complex regulatory interaction between RhlR and P. chlororaphis quorum-sensing signals and highlight the biotechnology potential of P. chlororaphis ATCC 9446 expressing P. aeruginosa rhlAB-R or rhlAB-R-C for the industrial production of RL.
Topics: Pseudomonas chlororaphis; Acyl-Butyrolactones; Pseudomonas aeruginosa; Soil; Bacterial Proteins; Glycolipids; Pseudomonas; 4-Butyrolactone
PubMed: 38041625
DOI: 10.1111/1751-7915.14377 -
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 -
Frontiers in Microbiology 2020PCL1606 (PcPCL1606) is a rhizobacterium isolated from avocado roots, which is a favorable niche for its development. This strain extensively interacts with plant roots...
PCL1606 (PcPCL1606) is a rhizobacterium isolated from avocado roots, which is a favorable niche for its development. This strain extensively interacts with plant roots and surrounding microbes and is considered a biocontrol rhizobacterium. Genome sequencing has shown the presence of thirty-one potential methyl-accepting chemotaxis proteins (MCPs). Among these MCPs, two candidates are putative functional aerotaxis receptors, encoded at locus PCL1606_41090 (1-1) and locus PLC1606_20530 (1-2), that are homologous to the Aer receptor of strain PaO1. Single- and double-deletion mutants in one or both genes have led to motility deficiencies in oxygen-rich areas, particularly reduced swimming motility compared with that of wildtype PcPCL1606. No differences in swarming tests were detected, and less adhesion by the double mutant was observed. However, the single and double mutants on avocado plant roots showed delayed biocontrol ability. During the first days of the biocontrol experiment, the -defective mutants also showed delayed root colonization. The current research characterizes the presence of transductors on . Thus, the functions of the PCL1606_41090 and PCL1606_20530 loci, corresponding to genes 1-1 and 1-2, respectively, are elucidated.
PubMed: 32754135
DOI: 10.3389/fmicb.2020.01560 -
Frontiers in Plant Science 2022(Pc) representatives are found as part of the rhizosphere-associated microbiome, and different rhizospheric Pc strains frequently perform beneficial activities for the...
(Pc) representatives are found as part of the rhizosphere-associated microbiome, and different rhizospheric Pc strains frequently perform beneficial activities for the plant. In this study we described the interactions between the rhizospheric Pc strains PCL1601, PCL1606 and PCL1607 with a focus on their effects on root performance. Differences among the three rhizospheric Pc strains selected were first observed in phylogenetic studies and confirmed by genome analysis, which showed variation in the presence of genes related to antifungal compounds or siderophore production, among others. Observation of the interactions among these strains under lab conditions revealed that PCL1606 has a better adaptation to environments rich in nutrients, and forms biofilms. Interaction experiments on plant roots confirmed the role of the different phenotypes in their lifestyle. The PCL1606 strain was the best adapted to the habitat of avocado roots, and PCL1607 was the least, and disappeared from the plant root scenario after a few days of interaction. These results confirm that 2 out 3 rhizospheric Pc strains were fully compatible (PCL1601 and PCL1606), efficiently colonizing avocado roots and showing biocontrol activity against the fungal pathogen . The third strain (PCL1607) has colonizing abilities when it is alone on the root but displayed difficulties under the competition scenario, and did not cause deleterious effects on the other Pc competitors when they were present. These results suggest that strains PCL1601 and PCL1606 are very well adapted to the avocado root environment and could constitute a basis for constructing a more complex beneficial microbial synthetic community associated with avocado plant roots.
PubMed: 36589057
DOI: 10.3389/fpls.2022.1063182 -
Frontiers in Microbiology 2024The rhizosphere microbiome is critical to plant health and resistance. PGPR are well known as plant-beneficial bacteria and generally regulate nutrient utilization as...
INTRODUCTION
The rhizosphere microbiome is critical to plant health and resistance. PGPR are well known as plant-beneficial bacteria and generally regulate nutrient utilization as well as plant responses to environmental stimuli. In our previous work, one typical PGPR strain, IRHB3, isolated from the soybean rhizosphere, had positive impacts on soil-borne disease suppression and growth promotion in the greenhouse, but its biocontrol mechanism and application in the field are not unclear.
METHODS
In the current study, IRHB3 was introduced into field soil, and its effects on the local rhizosphere microbiome, disease resistance, and soybean growth were comprehensively analyzed through high-throughput sequencing and physiological and molecular methods.
RESULTS AND DISCUSSION
We found that IRHB3 significantly increased the richness of the bacterial community but not the structure of the soybean rhizosphere. Functional bacteria related to phosphorus solubilization and nitrogen fixation, such as , , , , and , were recruited in rich abundance by IRHB3 to the soybean rhizosphere as compared to those without IRHB3. In addition, the IRHB3 supplement obviously maintained the homeostasis of the rhizosphere microbiome that was disturbed by , resulting in a lower disease index of root rot when compared with . Furthermore, JA-mediated induced resistance was rapidly activated by IRHB3 following and expression, and meanwhile, a set of nodulation genes, , , and , were also considerably induced by IRHB3 to improve nitrogen fixation ability and promote soybean yield, even when plants were infected by . Thus, IRHB3 tends to synergistically interact with local rhizosphere microbes to promote host growth and induce host resistance in the field.
PubMed: 38380096
DOI: 10.3389/fmicb.2024.1328863 -
JACS Au Nov 2023Chemical fertilizers have been crucial for sustaining the current global population by supplementing overused farmland to support consistent food production, but their...
Chemical fertilizers have been crucial for sustaining the current global population by supplementing overused farmland to support consistent food production, but their use is unsustainable. is a nitrogen-fixing bacterium that could be used as a fertilizer replacement, but this microbe is delicate. It is sensitive to stressors, such as freeze-drying and high temperatures. Here, we demonstrate protection of from freeze-drying, high temperatures (50 C), and high humidity using self-assembling metal-phenolic network (MPN) coatings. The composition of the MPN is found to significantly impact its protective efficacy, and with optimized compositions, no viability loss is observed for MPN-coated microbes under conditions where uncoated cells do not survive. Further, we demonstrate that MPN-coated microbes improve germination of seeds by 150% as compared to those treated with fresh . Taken together, these results demonstrate the protective capabilities of MPNs against environmental stressors and represent a critical step towards enabling the production and storage of delicate microbes under nonideal conditions.
PubMed: 38034965
DOI: 10.1021/jacsau.3c00426 -
Plants (Basel, Switzerland) Sep 2023The role of Calcium ions (Ca) is extensively documented and comprehensively understood in eukaryotic organisms. Nevertheless, emerging insights, primarily derived from... (Review)
Review
The role of Calcium ions (Ca) is extensively documented and comprehensively understood in eukaryotic organisms. Nevertheless, emerging insights, primarily derived from studies on human pathogenic bacteria, suggest that this ion also plays a pivotal role in prokaryotes. In this review, our primary focus will be on unraveling the intricate Ca toolkit within prokaryotic organisms, with particular emphasis on its implications for plant growth-promoting rhizobacteria (PGPR). We undertook an in silico exploration to pinpoint and identify some of the proteins described in the existing literature, including prokaryotic Ca channels, pumps, and exchangers that are responsible for regulating intracellular Calcium concentration ([Ca]), along with the Calcium-binding proteins (CaBPs) that play a pivotal role in sensing and transducing this essential cation. These investigations were conducted in four distinct PGPR strains: subsp. SMMP3, SVBP6, sp. BP01, and sp. 2A, which have been isolated and characterized within our research laboratories. We also present preliminary experimental data to evaluate the influence of exogenous Ca concentrations ([Ca]) on the growth dynamics of these strains.
PubMed: 37836138
DOI: 10.3390/plants12193398 -
Biology Feb 2022Endophenazine A is a terpenoid phenazine with phenazine-1-carboxylic acid (PCA), and dimethylallyl diphosphate (DMAPP) derived from the 2-methyl-D-erythritol-4-phosphate...
Endophenazine A is a terpenoid phenazine with phenazine-1-carboxylic acid (PCA), and dimethylallyl diphosphate (DMAPP) derived from the 2-methyl-D-erythritol-4-phosphate (MEP) pathway as the precursor, which shows good antimicrobial activity against several Gram-positive bacteria and fungi. However, the highest yield of endophenazine A was about 20 mg/L in , limiting its large-scale industrial development. P3, possessing an efficient PCA synthesis and MEP pathways, is a suitable chassis to synthesize endophenazine A. Herein, we designed an artificial biosynthetic pathway for the synthesis of endophenazine A in P3. Primarily, the prenyltransferase PpzP from 9663 was introduced into P3 and successfully synthesized endophenazine A. Another phenazine compound, endophenazine A1, was discovered and identified as a leakage of the intermediate 4-hydroxy-3-methyl-2-butene pyrophosphate (HMBPP). Finally, the yield of endophenazine A reached 279.43 mg/L, and the yield of endophenazine A1 reached 189.2 mg/L by metabolic engineering and medium optimization. In conclusion, we successfully synthesized endophenazine A and endophenazine A1 in P3 for the first time and achieved the highest titer, which provides a reference for the heterologous synthesis of terpenoid phenazines.
PubMed: 35336738
DOI: 10.3390/biology11030363 -
Microbial Cell Factories May 20202-Acetamidophenol (AAP) is an aromatic compound with the potential for antifungal, anti-inflammatory, antitumor, anti-platelet, and anti-arthritic activities. Due to the...
BACKGROUND
2-Acetamidophenol (AAP) is an aromatic compound with the potential for antifungal, anti-inflammatory, antitumor, anti-platelet, and anti-arthritic activities. Due to the biosynthesis of AAP is not yet fully understood, AAP is mainly produced by chemical synthesis. Currently, metabolic engineering of natural microbial pathway to produce valuable aromatic compound has remarkable advantages and exhibits attractive potential. Thus, it is of paramount importance to develop a dominant strain to produce AAP by elucidating the AAP biosynthesis pathway.
RESULT
In this study, the active aromatic compound AAP was first purified and identified in gene phzB disruption strain HT66ΔphzB, which was derived from Pseudomonas chlororaphis HT66. The titer of AAP in the strain HT66ΔphzB was 236.89 mg/L. Then, the genes involved in AAP biosynthesis were determined. Through the deletion of genes phzF, Nat and trpE, AAP was confirmed to have the same biosynthesis route as phenazine-1-carboxylic (PCA). Moreover, a new arylamine N-acetyltransferases (NATs) was identified and proved to be the key enzyme required for generating AAP by in vitro assay. P. chlororaphis P3, a chemical mutagenesis mutant strain of HT66, has been demonstrated to have a robust ability to produce antimicrobial phenazines. Therefore, genetic engineering, precursor addition, and culture optimization strategies were used to enhance AAP production in P. chlororaphis P3. The inactivation of phzB in P3 increased AAP production by 92.4%. Disrupting the phenazine negative regulatory genes lon and rsmE and blocking the competitive pathway gene pykA in P3 increased AAP production 2.08-fold, which also confirmed that AAP has the same biosynthesis route as PCA. Furthermore, adding 2-amidophenol to the KB medium increased AAP production by 64.6%, which suggested that 2-amidophenol is the precursor of AAP. Finally, by adding 5 mM 2-amidophenol and 2 mM Fe to the KB medium, the production of AAP reached 1209.58 mg/L in the engineered strain P3ΔphzBΔlonΔpykAΔrsmE using a shaking-flask culture. This is the highest microbial-based AAP production achieved to date.
CONCLUSION
In conclusion, this study clarified the biosynthesis process of AAP in Pseudomonas and provided a promising host for industrial-scale biosynthesis of AAP from renewable resources.
Topics: Acetaminophen; Arylamine N-Acetyltransferase; Bacterial Proteins; Biosynthetic Pathways; Genes, Bacterial; Industrial Microbiology; Metabolic Engineering; Pseudomonas chlororaphis
PubMed: 32430011
DOI: 10.1186/s12934-020-01364-7 -
Scientific Reports Aug 2021Trans-2,3-dihydro-3-hydroxyanthranilic acid (DHHA) is a cyclic β-amino acid used for the synthesis of non-natural peptides and chiral materials. And it is an...
Trans-2,3-dihydro-3-hydroxyanthranilic acid (DHHA) is a cyclic β-amino acid used for the synthesis of non-natural peptides and chiral materials. And it is an intermediate product of phenazine production in Pseudomonas spp. Lzh-T5 is a P. chlororaphis strain isolated from tomato rhizosphere found in China. It can synthesize three antifungal phenazine compounds. Disruption the phzF gene of P. chlororaphis Lzh-T5 results in DHHA accumulation. Several strategies were used to improve production of DHHA: enhancing the shikimate pathway by overexpression, knocking out negative regulatory genes, and adding metal ions to the medium. In this study, three regulatory genes (psrA, pykF, and rpeA) were disrupted in the genome of P. chlororaphis Lzh-T5, yielding 5.52 g/L of DHHA. When six key genes selected from the shikimate, pentose phosphate, and gluconeogenesis pathways were overexpressed, the yield of DHHA increased to 7.89 g/L. Lastly, a different concentration of Fe was added to the medium for DHHA fermentation. This genetically engineered strain increased the DHHA production to 10.45 g/L. According to our result, P. chlororaphis Lzh-T5 could be modified as a microbial factory to produce DHHA. This study laid a good foundation for the future industrial production and application of DHHA.
Topics: 3-Hydroxyanthranilic Acid; Culture Media; Fermentation; Ferric Compounds; Gene Knockdown Techniques; Genes, Bacterial; Genes, Regulator; Phenazines; Pseudomonas chlororaphis
PubMed: 34385485
DOI: 10.1038/s41598-021-94674-8