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Metabolic Engineering Jul 2023The emergence of next-generation sequencing (NGS) technologies has made it possible to not only sequence entire genomes, but also identify metabolic engineering targets...
The emergence of next-generation sequencing (NGS) technologies has made it possible to not only sequence entire genomes, but also identify metabolic engineering targets across the pangenome of a microbial population. This study leverages NGS data as well as existing molecular biology and bioinformatics tools to identify and validate genomic signatures for improving phenazine biosynthesis in Pseudomonas chlororaphis. We sequenced a diverse collection of 34 Pseudomonas isolates using short- and long-read sequencing techniques and assembled whole genomes using the NGS reads. In addition, we assayed three industrially relevant phenotypes (phenazine production, biofilm formation, and growth temperature) for these isolates in two different media conditions. We then provided the whole genomes and phenazine production data to a unitig-based microbial genome-wide association study (mGWAS) tool to identify novel genomic signatures responsible for phenazine production in P. chlororaphis. Post-processing of the mGWAS analysis results yielded 330 significant hits influencing the biosynthesis of one or more phenazine compounds. Based on a quantitative metric (called the phenotype score), we elucidated the most influential hits for phenazine production and experimentally validated them in vivo in the most optimal phenazine producing strain. Two genes significantly increased phenazine-1-carboxamide (PCN) production: a histidine transporter (ProY_1), and a putative carboxypeptidase (PS__04251). A putative MarR-family transcriptional regulator decreased PCN titer when overexpressed in a high PCN producing isolate. Overall, this work seeks to demonstrate the utility of a population genomics approach as an effective strategy in enabling the identification of targets for metabolic engineering of bioproduction hosts.
Topics: Pseudomonas chlororaphis; Metagenomics; Genome-Wide Association Study; Pseudomonas; Phenazines; Bacterial Proteins
PubMed: 37369325
DOI: 10.1016/j.ymben.2023.06.008 -
3 Biotech Jun 2023The recent advances in omics and computational analysis have enabled the capacity to identify the exclusive strain-specific metabolites and novel biosynthetic gene...
UNLABELLED
The recent advances in omics and computational analysis have enabled the capacity to identify the exclusive strain-specific metabolites and novel biosynthetic gene clusters. This study analyzed eight strains of including GS1, GS3, GS4, GS6, GS7, FS2, ARS38, PBSt2, one strain of RP4, one strain of (At1RP4), and one strain of (RS1) for the production of rhamnolipids, quorum-sensing signals, and osmolytes. Seven rhamnolipid derivatives were variably detected in fluorescent pseudomonads. These rhamnolipids included Rha-C-C, Rha-Rha-C-C, Rha-C-Cdb, Rha-C-C, Rha-Rha-C-C Rha-C-C, and Rha-Rha-C-Cdb. spp. also showed the variable production of osmoprotectants including N-acetyl glutaminyl glutamine amide (NAGGN), betaine, ectoine, and trehalose. Betaine and ectoine were produced by all pseudomonads, however, NAGGN and trehalose were observed by five and three strains, respectively. Four strains including (RP4), (At1RP4), (RS1), and (PBSt2) were exposed to 1- 4% NaCl concentrations and evaluated for the changes in phenazine production profile which were negligible. AntiSMASH 5.0 platform showed 50 biosynthetic gene clusters in PB-St2, of which 23 (45%) were classified as putative gene clusters with ClusterFinder algorithm, five (10%) were classified as non-ribosomal peptides synthetases (NRPS), five (10%) as saccharides, and four (8%) were classified as putative fatty acids. The genomic attributes and comprehensive insights into the metabolomic profile of these spp. strains showcase their phytostimulatory, phyto-protective, and osmoprotective effects of diverse crops grown in normal and saline soils.
SUPPLEMENTARY INFORMATION
The online version contains supplementary material available at 10.1007/s13205-023-03607-x.
PubMed: 37193329
DOI: 10.1007/s13205-023-03607-x -
Frontiers in Microbiology 2023Phenazine-1-carboxylic acid (PCA) is a biologically active substance with the ability to prevent and control crop diseases. It was certified as a pesticide by the...
Phenazine-1-carboxylic acid (PCA) is a biologically active substance with the ability to prevent and control crop diseases. It was certified as a pesticide by the Ministry of Agriculture of China in 2011 and was named "Shenzimycin." Lzh-T5 is a strain found in the rhizosphere of tomatoes. This strain can produce only 230 mg/L of PCA. We used LDA-4, which produces the phenazine synthetic intermediate trans-2,3-dihydro-3-hydroxyanthranilic acid in high amounts, as the starting strain. By restoring and knocking out , we achieved PCA accumulation. Moreover, PCA production was enhanced after knocking out negative regulators, enhancing the shikimate pathway, and performing fed-batch fermentation, thus resulting in the production of 10,653 mg/L of PCA. It suggested that Lzh-T5 has the potential to become an efficiency cell factory of biologically active substances.
PubMed: 37168109
DOI: 10.3389/fmicb.2023.1186052 -
Frontiers in Microbiology 2023The jumbo phages encode proteins that assemble to form a nucleus-like compartment in infected cells. Here we report the cryo-EM structure and biochemistry...
The jumbo phages encode proteins that assemble to form a nucleus-like compartment in infected cells. Here we report the cryo-EM structure and biochemistry characterization of gp105, a protein that is encoded by the jumbo phage 201φ2-1 and is involved in the formation of the nucleus-like compartment in phage 201φ2-1 infected . We found that, although most gp105 molecules are in the monomeric state in solution, a small portion of gp105 assemble to form large sheet-like assemblies and small cube-like particles. Reconstruction of the cube-like particles showed that the particle consists of six flat head-to-tail tetramers arranged into an octahedral cube. The four molecules at the contact interface of two head-to-tail tetramers are 2-fold symmetry-related and constitute a concave tetramer. Further reconstructions without applying symmetry showed that molecules in the particles around the distal ends of a 3-fold axis are highly dynamic and have the tendency to open up the assembly. Local classifications and refinements of the concave tetramers in the cube-like particle resulted in a map of the concave tetramer at a resolution of 4.09 Å. Structural analysis of the concave tetramer indicates that the N and C terminal fragments of gp105 are important for mediating the intermolecular interactions, which was further confirmed by mutagenesis studies. Biochemistry assays showed that, in solution, the cube-like particles of gp105 are liable to either disassemble to form the monomers or recruit more molecules to form the high molecular weight lattice-like assembly. We also found that monomeric gp105s can self-assemble to form large sheet-like assemblies , and the assembly of gp105 is a reversible dynamic process and temperature-dependent. Taken together, our results revealed the dynamic assembly of gp105, which helps to understand the development and function of the nucleus-like compartment assembled by phage-encoded proteins.
PubMed: 37138628
DOI: 10.3389/fmicb.2023.1170112 -
Microbiology Spectrum Mar 2023Secretion of exoproteins is a key component of bacterial virulence, and is tightly regulated in response to environmental stimuli and host-dependent signals. The...
Secretion of exoproteins is a key component of bacterial virulence, and is tightly regulated in response to environmental stimuli and host-dependent signals. The entomopathogenic bacterium Yersinia entomophaga MH96 produces a wide range of exoproteins including its main virulence factor, the 2.46 MDa insecticidal Yen-Tc toxin complex. Previously, a high-throughput transposon-based screening assay identified the region of exoprotein release (YeRER) as essential to exoprotein release in MH96. This study defines the role of the YeRER associated ambiguous holin/endolysin-based lysis cluster (ALC) and the novel RoeA regulator in the regulation and release of exoproteins in MH96. A mutation in the ambiguous lysis cassette (ALC) region abolished exoprotein release and caused cell elongation, a phenotype able to be restored through -complementation with an intact ALC region. Endogenous ALC did not impact cell growth of the wild type, while artificial expression of an optimized ALC caused cell lysis. Using HolA-sfGFP and Rz1-sfGFP reporters, Rz1 expression was observed in all cells while HolA expression was limited to a small proportion of cells, which increased over time. Transcriptomic assessments found expression of the genes encoding the prominent exoproteins, including the Yen-Tc, was reduced in the mutant and identified a 220 ncRNA of the YeRER intergenic region that, when complemented in the wildtype, abolished exoprotein release. A model for mediated exoprotein regulation and release is proposed. While theoretical models exist, there is not yet any empirical data that links ALC phage-like lysis cassettes with the release of large macro-molecular toxin complexes, such as Yen-Tc in Gram-negative bacteria. In this study, we demonstrate that the novel RoeA activates the production of exoproteins (including Yen-Tc) and the ALC at the transcriptional level. The translation of the ALC holin is confined to a subpopulation of cells that then lyse over time, indicative of a complex hierarchical regulatory network. The presence of an orthologous RoeA and a HolA like holin 5' of an eCIS Afp element in Pseudomonas chlororaphis, combined with the presented data, suggests a shared mechanism is required for the release of some large macromolecular protein assemblies, such as the Yen-Tc, and further supports classification of phage-like lysis clusters as type 10 secretion systems.
PubMed: 36951587
DOI: 10.1128/spectrum.00364-23 -
Microbiology Spectrum Mar 2023Within bacterial communities, community members engage in interactions employing diverse offensive and defensive tools to reach coexistence. Extracellular-matrix...
Within bacterial communities, community members engage in interactions employing diverse offensive and defensive tools to reach coexistence. Extracellular-matrix production and sporulation are defensive mechanisms used by Bacillus subtilis cells when they interact with Pseudomonas chlororaphis strains expressing a type VI secretion system (T6SS). Here, we define Tse1 as the main toxin mobilized by the Pseudomonas chlororaphis T6SS that triggers sporulation in Bacillus subtilis. We characterize Tse1 as a peptidoglycan hydrolase that indirectly alters the dynamics and functionality of the cell membrane. We also delineate the response of cells to Tse1, which through the coordinated actions of the extracellular sigma factor σ and the cytoplasmic histidine kinases KinA and KinB, culminates in activation of the sporulation cascade. We propose that this cellular developmental response permits bacilli to defend against the toxicity of T6SS-mobilized Tse1 effector. The study of bacterial interactions is helping to define species-specific strategies used to modulate the competition dynamics underlying the development of community compositions. In this study, we deciphered the role of Pseudomonas T6SS when competing with and the mechanism by which a T6SS-toxin modifies physiology. We found that Pseudomonas triggers sporulation by injecting through T6SS a toxin that we called Tse1. We found that Tse1 is a hydrolase that degrades peptidoglycan and indirectly damages membrane functionality. In addition, we demonstrated the mechanism by which cells increase the sporulation rate upon recognition of the presence of Tse1. Interestingly, asporogenic cells are more sensitive to T6SS activity, which led us to propose sporulation as a last resort of bacilli to overcome this family of toxins.
PubMed: 36916921
DOI: 10.1128/spectrum.05045-22 -
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 -
Microbial Ecology Oct 2023Combining different biocontrol agents (BCA) is an approach to increase efficacy and reliability of biological control. If several BCA are applied together, they have to...
Combining different biocontrol agents (BCA) is an approach to increase efficacy and reliability of biological control. If several BCA are applied together, they have to be compatible and ideally work together. We studied the interaction of a previously selected BCA consortium of entomopathogenic pseudomonads (Pseudomonas chlororaphis), nematodes (Steinernema feltiae associated with Xenorhabdus bovienii), and fungi (Metarhizium brunneum). We monitored the infection course in a leaf- (Pieris brassicae) and a root-feeding (Diabrotica balteata) pest insect after simultaneous application of the three BCA as well as their interactions inside the larvae in a laboratory setting. The triple combination caused the highest mortality and increased killing speed compared to single applications against both pests. Improved efficacy against P. brassicae was mainly caused by the pseudomonad-nematode combination, whereas the nematode-fungus combination accelerated killing of D. balteata. Co-monitoring of the three BCA and the nematode-associated Xenorhabdus symbionts revealed that the four organisms are able to co-infect the same larva. However, with advancing decay of the cadaver there is increasing competition and cadaver colonization is clearly dominated by the pseudomonads, which are known for their high competitivity in the plant rhizosphere. Altogether, the combination of the three BCA increased killing efficacy against a Coleopteran and a Lepidopteran pest which indicates that this consortium could be applied successfully against a variety of insect pests.
Topics: Animals; Reproducibility of Results; Pest Control, Biological; Insecta; Larva; Rhabditida; Plant Leaves
PubMed: 36849610
DOI: 10.1007/s00248-023-02191-0 -
Applied and Environmental Microbiology Mar 2023IPD072Aa from Pseudomonas chlororaphis is a new insecticidal protein that has been shown to have high activity against western corn rootworm (WCR). IPD072 has no...
IPD072Aa from Pseudomonas chlororaphis is a new insecticidal protein that has been shown to have high activity against western corn rootworm (WCR). IPD072 has no sequence signatures or predicted structural motifs with any known protein revealing little insight into its mode of action using bioinformatic tools. As many bacterially derived insecticidal proteins are known to act through mechanisms that lead to death of midgut cells, we evaluated whether IPD072Aa also acts by targeting the cells of WCR midgut. IPD072Aa exhibits specific binding to brush border membrane vesicles (BBMVs) prepared from WCR guts. The binding was found to occur at binding sites that are different than those recognized by Cry3A or Cry34Ab1/Cry35Ab1, proteins expressed by current maize traits that target WCR. Using fluorescence confocal microscopy, immuno-detection of IPD072Aa in longitudinal sections from whole WCR larvae that were fed IPD072Aa revealed the association of the protein with the cells that line the gut. High-resolution scanning electron microscopy of similar whole larval sections revealed the disruption of the gut lining resulting from cell death caused by IPD072Aa exposure. These data show that the insecticidal activity of IPD072Aa results from specific targeting and killing of rootworm midgut cells. Transgenic traits targeting WCR based on insecticidal proteins from Bacillus thuringiensis have proven effective in protecting maize yield in North America. High adoption has led to WCR populations that are resistant to the trait proteins. Four proteins have been developed into commercial traits, but they represent only two modes of action due to cross-resistance among three. New proteins suited for trait development are needed. IPD072Aa, identified from the bacterium Pseudomonas chlororaphis, was shown to be effective in protecting transgenic maize against WCR. To be useful, IPD072Aa must work through binding to different receptors than those utilized by current traits to reduce risk of cross-resistance and understanding its mechanism of toxicity could aid in countering resistance development. Our results show that IPD072Aa binds to receptors in WCR gut that are different than those utilized by current commercial traits and its targeted killing of midgut cells results in larval death.
Topics: Animals; Zea mays; Pseudomonas chlororaphis; Endotoxins; Coleoptera; Larva; Bacillus thuringiensis; Insecticides; Bacterial Proteins; Epithelial Cells; Plants, Genetically Modified; Pest Control, Biological
PubMed: 36847510
DOI: 10.1128/aem.01622-22 -
Frontiers in Microbiology 2023The spectral distribution of light (different wavelength) has recently been identified as an important factor in the dynamics and function of leaf-associated microbes....
BACKGROUND
The spectral distribution of light (different wavelength) has recently been identified as an important factor in the dynamics and function of leaf-associated microbes. This study investigated the impact of different wavelength on three commercial biocontrol agents (BCA): (BA), (PC), and (SG).
METHODS
The impact of light exposure on sole carbon source utilization, biofilm formation, and biosurfactant production by the selected BCA was studied using phenotypic microarray (PM) including 190 sole carbon sources (OmniLog®, PM panels 1 and 2). The BCA were exposed to five monochromatic light conditions (420, 460, 530, 630, and 660 nm) and darkness during incubation, at an intensity of 50 μmol m s.
RESULTS
Light exposure together with specific carbon source increased respiration in all three BCA. Different wavelengths of light influenced sole carbon utilization for the different BCA, with BA and PC showing increased respiration when exposed to wavelengths within the blue spectrum (420 and 460 nm) while respiration of selected carbon sources by SG increased in the presence of red light (630 and 660 nm). Only one carbon source (capric acid) generated biosurfactant production in all three BCA. A combination of specific wavelength of light and sole carbon source increased biofilm formation in all three BCA. BA showed significantly higher biofilm formation when exposed to blue (460 nm) and green (530 nm) light and propagated in D-sucrose, D-fructose, and dulcitol. PC showed higher biofilm formation when exposed to blue light. Biofilm formation by SG increased when exposed to red light (630 nm) and propagated in citraconic acid.
CONCLUSION
To increase attachment and success in BCA introduced into the phyllosphere, a suitable combination of light quality and nutrient conditions could be used.
PubMed: 36819051
DOI: 10.3389/fmicb.2023.1087639