Indirect Exposure to Atrocities and Post-Traumatic Stress Disorder Symptoms among Aid Workers: Hemispheric Lateralization Matters.

Background: Humanitarian aid workers (HAWs) are indirectly exposed to atrocities relating to people of concern (POC). This may result in a risk of secondary traumatization demonstrated by post-traumatic stress symptoms (PTSSs). Previous studies have demonstrated that hemispheric lateralization (HL) moderates the relationship between threat exposure and post-traumatic stress symptoms (PTSSs). Aims: We hypothesized that indirect exposure to atrocities (IETA) would be positively correlated with PTSSs among HAWs with right and not left HL. Method: Fifty-four HAWs from several countries that provided humanitarian support in Greece and Colombia participated in this correlational and cross-sectional observation study. They completed scales relating to IETA, PTSSs were assessed using a brief, valid scale, and HL was measured. Results: IETA was positively and significantly related to PTSSs (r = 0.39, p < 0.005). Considering HL, IETA was unrelated to PTSSs among people with right HL (r = 0.29, p = 0.14), while IETA was related to PTSSs among people with left HL (r = 0.52, p = 0.008). Right HL emerged as a protective factor in the relationship between IETA and PTSS. Conclusions: An assessment of dominant HL can serve as one consideration among others when deploying HAWs in specific locations and roles, vis à vis IETA. Moreover, those found to have a higher risk for PTSSs based on their HL could be monitored more closely to prevent adverse reactions to IETA.

Biosynthesis and engineering of the nonribosomal peptides with a C-terminal putrescine.

The broad bioactivities of nonribosomal peptides rely on increasing structural diversity. Genome mining of the Burkholderiales strain Schlegelella brevitalea DSM 7029 leads to the identification of a class of dodecapeptides, glidonins, that feature diverse N-terminal modifications and a uniform putrescine moiety at the C-terminus. The N-terminal diversity originates from the wide substrate selectivity of the initiation module. The C-terminal putrescine moiety is introduced by the unusual termination module 13, the condensation domain directly catalyzes the assembly of putrescine into the peptidyl backbone, and other domains are essential for stabilizing the protein structure. Swapping of this module to another two nonribosomal peptide synthetases leads to the addition of a putrescine to the C-terminus of related nonribosomal peptides, improving their hydrophilicity and bioactivity. This study elucidates the mechanism for putrescine addition and provides further insights to generate diverse and improved nonribosomal peptides by introducing a C-terminal putrescine.

The Genomic-Driven Discovery of Glutarimide-Containing Derivatives from Burkholderia gladioli.

Glutarimide-containing polyketides exhibiting potent antitumor and antimicrobial activities were encoded via conserved module blocks in various strains that favor the genomic mining of these family compounds. The bioinformatic analysis of the genome of Burkholderia gladioli ATCC 10248 showed a silent trans-AT PKS biosynthetic gene cluster (BGC) on chromosome 2 (Chr2C8), which was predicted to produce new glutarimide-containing derivatives. Then, the silent polyketide synthase gene cluster was successfully activated via in situ promoter insertion and heterologous expression. As a result, seven glutarimide-containing analogs, including five new ones, gladiofungins D-H (3-7), and two known gladiofungin A/gladiostatin (1) and 2 (named gladiofungin C), were isolated from the fermentation of the activated mutant. Their structures were elucidated through the analysis of HR-ESI-MS and NMR spectroscopy. The structural diversities of gladiofungins may be due to the degradation of the butenolide group in gladiofungin A (1) during the fermentation and extraction process. Bioactivity screening showed that 2 and 4 had moderate anti-inflammatory activities. Thus, genome mining combined with promoter engineering and heterologous expression were proved to be effective strategies for the pathway-specific activation of the silent BGCs for the directional discovery of new natural products.

Heterologous Biosynthesis of Complex Bacterial Natural Products in Burkholderia gladioli.

Bacterial natural products (NPs) are an indispensable source of drugs and biopesticides. Heterologous expression is an essential method for discovering bacterial NPs and the efficient biosynthesis of valuable NPs, but the chassis for Gram-negative bacterial NPs remains inadequate. In this study, we built a Burkholderiales mutant Burkholderia gladioli Δgbn::attB by introducing an integrated site (attB) to inactivate the native gladiolin (gbn) biosynthetic gene cluster, which stabilizes large foreign gene clusters and reduces the native metabolite profile. The growth and successful heterologous production of high-value NPs such as phylogenetically close Burkholderiales-derived antitumor polyketides (PKs) rhizoxins, phylogenetically distant Gammaproteobacteria-derived anti-MRSA (methicillin-resistant Staphylococcus aureus) antibiotics WAP-8294As, and Deltaproteobacteria-derived antitumor PKs disorazols demonstrate that this strain is a potential chassis for Gram-negative bacterial NPs. We further improved the yields of WAP-8294As through promoter insertions and precursor pathway overexpression based on heterologous expression in this strain. This study provides a robust bacterial chassis for genome mining, efficient production, and molecular engineering of bacterial NPs.

Primary Intracranial Hemorrhage: Characteristics, Distribution, Risk Factors, and Outcomes-A Comparative Study between Jewish and Arab Ethnic Groups in Northern Israel.

Background and purpose: This study aimed to investigate the differences in intracerebral hemorrhage (ICH) between Jews and Arabs residing in northern Israel, focusing on risk factors, hemorrhage volume, and functional outcome. Methods: A retrospective analysis was conducted utilizing a population-based registry to investigate intracerebral hemorrhage (ICH) characteristics, risk factors, and outcomes. The registry consisted of inpatients diagnosed with hemorrhagic stroke. Due to the wide variation in data on ICH characteristics and the limited availability of population-based data on predictors of ICH survival and functional outcomes, we collected retrospective data on all adult patients admitted to the Galilee Medical Center with a diagnosis of ICH. Data were obtained from the registry covering the period from 2013 to 2019. Ethnic differences and risk factors associated with intracranial hemorrhage (ICH) were examined within a diverse population of 241 patients, comprising 52.70% Jews (n = 127) and 47.30% Arabs (n = 114). Results: The results of this study revealed significant differences in age, obesity rates, and intracerebral hemorrhage (ICH) location between the two ethnic groups. Hypertension emerged as the most prevalent condition among ICH patients in both ethnic groups (76.70%), followed primarily by anticoagulant use (63.60%), dyslipidemia (60.70%), diabetes (44.60%), obesity (30.60%), smoking (24.60%), and a history of cardiovascular disease (21.80%). Furthermore, 20.90% of the patients had a history of previous cerebrovascular accidents (CVA). Arab patients with ICH were generally younger (62.90 ± 16.00 years) and exhibited higher rates of obesity (38.70%) compared to Jewish patients with ICH (70.17 ± 15.24 years, 23% obesity; p = 0.001, p = 0.013, respectively). Hemorrhage volume was identified as a crucial determinant of patient outcomes, with larger volumes associated with poorer Modified Rankin Scale (mRS) scores at discharge and higher mortality rates. Interestingly, patients without hypertension had higher hemorrhage volumes compared to those with hypertension. The extent of hemorrhage into the ventricles did not significantly correlate with mRS at discharge in our dataset. Conclusions: This study highlights significant differences in the characteristics and outcomes of intracranial hemorrhage (ICH) between Jews and Arabs in northern Israel. The findings reveal variations in age, obesity rates, and ICH location between the two groups. While hypertension was the most prevalent risk factor for both populations, other risk factors differed. Notably, hemorrhage volume emerged as a crucial prognostic factor, aligning with previously published data. These findings underscore the necessity for tailored approaches that consider ethnic-specific factors in the risk assessment, prevention, and management of ICH. Further research is warranted to elucidate the underlying mechanisms and develop interventions aimed at improving outcomes and enhancing healthcare practices in ICH management.

Vagal Nerve Activity and Short-Term Clinical Outcomes after Stroke: What Is Left May Not Be Right.

Stroke is a leading cause of death worldwide. Multiple factors influence the severity of stroke. Normal functional and biological differences seen between the hemispheres may also be related to stroke severity. In the present study, we examined the differences in the severity of stroke as a function of stroke side, and whether patients' vagal nerve activity moderated such differences. We included 87 patients with an ischemic stroke, whose medical records were retrospectively examined for background information (age, gender), stroke side and severity by NIHSS, length of stay in hospital, inflammation such as C-reactive protein, and vagal nerve activity. The vagal activity was indexed by patients' heart-rate variability (HRV), fluctuations in the intervals between normal heartbeats, derived from patients' ECG. Results revealed that patients with left-side stroke had significantly worse NIHSS scores (10.6) than those with right-sided stroke (7.6, p < 0.05). However, when dividing the sample into those with low versus high HRV (at the median), only when HRV was low, did patients with left-side stroke have a worse NIHSS score (10.9) compared to those with right-sided stroke (6.5, p < 0.05). In contrast, no differences in stroke severity were seen between left stroke (10.2) and right stoke (8.7, p > 0.05), when HRV was high. These results tended to remain the same when statistically controlling for age effects, which was related to NIHSS, but not to the stroke side. These findings suggest that patients with left-sided stroke may have more severe strokes than those with right-sided ones, but that adequate vagal nerve activity may protect against such differences. Possible mechanisms and suggestions for future directions are provided.

A novel tumor-targeting strain of Xenorhabdus stockiae exhibits potent biological activities.

Xenorhabdus are symbionts of soil entomopathogenic nematodes of the genus Steinernema presenting two distinct forms in their life cycle, and can produce a broad range of bioactive compounds. In this study, a novel Xenorhabdus stockiae strain HN_xs01 was isolated from a soil sample via an entrapment method using Galleria melonella nematodes. The supernatants of strain HN_xs01 exhibited antimicrobial properties against Gram-negative and Gram-positive bacteria, and insecticidal properties against Helicoverpa armigera larvae, and antitumor properties as well. Moreover, three linear rhabdopeptides (1, 2 and 3) were identified from strain HN_xs01 using nuclear magnetic resonance analysis, which exhibited significant cytotoxic activity against the human epithelial carcinoma cell line A431 and the human chronic myelogenous leukemia cell line K562. Some bacteria have been reported to colonize the tumor region, and we determined that HN_xs01 could grow in tumor xenografts in this study. HN_xs01 invaded and replicated in B16 melanoma cells grafted into C57BL/6 mice, resulting in tumor inhibition. Moreover, strain HN_xs01 not only merely aggregated in the tumor environment, but also prevented pulmonary metastasis. It caused fragmentation of vessels and cell apoptosis, which contributed to its antitumor effect. In conclusion, X. stockiae HN_xs01 is a novel tumor-targeting strain with potential applications in medicinal and agricultural fields.

Biosynthesis of Glidomides and Elucidation of Different Mechanisms for Formation of ß-OH Amino Acid Building Blocks.

Nonribosomal peptide synthetases (NRPSs) can incorporate nonproteinogenic amino acids into peptidyl backbones to increase structural diversity. Genome mining of Schlegelella brevitalea led to the identification of a class of linear lipoheptapeptides, glidomides, featuring two unusual residues: threo-ß-OH-L-His and threo-ß-OH-D-Asp. The ß-hydroxylation of Asp and His is catalyzed by the nonheme FeII /α-ketoglutarate-dependent ß-hydroxylases GlmD and GlmF, respectively. GlmD independently catalyzes the hydroxylation of L-Asp to primarily produce threo-ß-OH-L-Asp on the thiolation domain, and then undergoes epimerization to form threo-ß-OH-D-Asp in the final products. However, ß-hydroxylation of His requires the concerted action of GlmF and the interface (I) domain, a novel condensation domain family clade. The key sites of I domain for interaction with GlmF were identified, suggesting that the mechanism for hydroxylation of His depends on the collaboration between hydroxylase and NRPS.

Mutant ANP induces mitochondrial and ion channel remodeling in a human iPSC-derived atrial fibrillation model.

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) can model heritable arrhythmias to personalize therapies for individual patients. Although atrial fibrillation (AF) is a leading cause of cardiovascular morbidity and mortality, current platforms to generate iPSC-atrial (a) CMs are inadequate for modeling AF. We applied a combinatorial engineering approach, which integrated multiple physiological cues, including metabolic conditioning and electrical stimulation, to generate mature iPSC-aCMs. Using the patient's own atrial tissue as a gold standard benchmark, we assessed the electrophysiological, structural, metabolic, and molecular maturation of iPSC-aCMs. Unbiased transcriptomic analysis and inference from gene regulatory networks identified key gene expression pathways and transcription factors mediating atrial development and maturation. Only mature iPSC-aCMs generated from patients with heritable AF carrying the non-ion channel gene (NPPA) mutation showed enhanced expression and function of a cardiac potassium channel and revealed mitochondrial electron transport chain dysfunction. Collectively, we propose that ion channel remodeling in conjunction with metabolic defects created an electrophysiological substrate for AF. Overall, our electro-metabolic approach generated mature human iPSC-aCMs that unmasked the underlying mechanism of the first non-ion channel gene, NPPA, that causes AF. Our maturation approach will allow for the investigation of the molecular underpinnings of heritable AF and the development of personalized therapies.

Enhanced growth of ginger plants by an eco- friendly nitrogen-fixing Pseudomonas protegens inoculant in glasshouse fields.

BACKGROUND: Excessive nitrogen (N) fertilization in glasshouse fields greatly increases N loss and fossil-fuel energy consumption resulting in serious environmental risks. Microbial inoculants are strongly emerging as potential alternatives to agrochemicals and offer an eco-friendly fertilization strategy to reduce our dependence on synthetic chemical fertilizers. Effects of a N-fixing strain Pseudomonas protegens CHA0-ΔretS-nif on ginger plant growth, yield, and nutrient uptake, and on earthworm biomass and the microbial community were investigated in glasshouse fields in Shandong Province, northern China. RESULTS: Application of CHA0-ΔretS-nif could promote ginger plant development, and significantly increased rhizome yields, by 12.93% and 7.09%, respectively, when compared to uninoculated plants and plants treated with the wild-type bacterial strain. Inoculation of CHA0-ΔretS-nif had little impact on plant phosphorus (P) acquisition, whereas it was associated with enhanced N and potassium (K) acquisition by ginger plants. Moreover, inoculation of CHA0-ΔretS-nif had positive effects on the bacteria population size and the number of earthworms in the rhizosphere. Similar enhanced performances were also found in CHA0-ΔretS-nif-inoculated ginger plants even when the N-fertilizer application rate was reduced by 15%. A chemical N input of 573.8 kg ha-1 with a ginger rhizome yield of 1.31 × 105 kg ha-1 was feasible. CONCLUSIONS: The combined application of CHA0-ΔretS-nif and a reduced level of N-fertilizers can be employed in glasshouse ginger production for the purpose of achieving high yields while at the same time reducing the inorganic-N pollution from traditional farming practices. © 2021 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Rational construction of genome-reduced Burkholderiales chassis facilitates efficient heterologous production of natural products from proteobacteria.

Heterologous expression of biosynthetic gene clusters (BGCs) avails yield improvements and mining of natural products, but it is limited by lacking of more efficient Gram-negative chassis. The proteobacterium Schlegelella brevitalea DSM 7029 exhibits potential for heterologous BGC expression, but its cells undergo early autolysis, hindering further applications. Herein, we rationally construct DC and DT series genome-reduced S. brevitalea mutants by sequential deletions of endogenous BGCs and the nonessential genomic regions, respectively. The DC5 to DC7 mutants affect growth, while the DT series mutants show improved growth characteristics with alleviated cell autolysis. The yield improvements of six proteobacterial natural products and successful identification of chitinimides from Chitinimonas koreensis via heterologous expression in DT mutants demonstrate their superiority to wild-type DSM 7029 and two commonly used Gram-negative chassis Escherichia coli and Pseudomonas putida. Our study expands the panel of Gram-negative chassis and facilitates the discovery of natural products by heterologous expression.

Development and application of an efficient recombineering system for Burkholderia glumae and Burkholderia plantarii.

The lambda phage Red proteins Redα/Redß/Redγ and Rac prophage RecE/RecT proteins are powerful tools for precise and efficient genetic manipulation but have been limited to only a few prokaryotes. Here, we report the development and application of a new recombineering system for Burkholderia glumae and Burkholderia plantarii based on three Rac bacteriophage RecET-like operons, RecETheBDU8 , RecEThTJI49 and RecETh1h2eYI23 , which were obtained from three different Burkholderia species. Recombineering experiments indicated that RecEThTJI49 and RecETh1h2eYI23 showed higher recombination efficiency compared to RecETheBDU8 in Burkholderia glumae PG1. Furthermore, all of the proteins currently categorized as hypothetical proteins in RecETh1h2eYI23, RecEThTJI49 and RecETheBDU8 may have a positive effect on recombination in B. glumae PG1 except for the h2 protein in RecETh1h2eYI23 . Additionally, RecETYI23 combined with exonuclease inhibitors Pluγ or Redγ exhibited equivalent recombination efficiency compared to Redγßα in Escherichia coli, providing potential opportunity of recombineering in other Gram-negative bacteria for its loose host specificity. Using recombinase-assisted in situ insertion of promoters, we successfully activated three cryptic non-ribosomal peptide synthetase biosynthetic gene clusters in Burkholderia strains, resulting in the generation of a series of lipopeptides that were further purified and characterized. Compound 7 exhibited significant potential anti-inflammatory activity by inhibiting lipopolysaccharide-stimulated nitric oxide production in RAW 264.7 macrophages. This recombineering system may greatly enhance functional genome research and the mining of novel natural products in the other species of the genus Burkholderia after optimization of a protocol.

Human induced pluripotent stem cell-derived atrial cardiomyocytes carrying an SCN5A mutation identify nitric oxide signaling as a mediator of atrial fibrillation.

Mutations in SCN5A, encoding the cardiac sodium channel, are linked with familial atrial fibrillation (AF) but the underlying pathophysiologic mechanisms and implications for therapy remain unclear. To characterize the pathogenesis of AF-linked SCN5A mutations, we generated patient-specific induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-aCMs) from two kindreds carrying SCN5A mutations (E428K and N470K) and isogenic controls using CRISPR-Cas9 gene editing. We showed that mutant AF iPSC-aCMs exhibited spontaneous arrhythmogenic activity with beat-to-beat irregularity, prolonged action potential duration, and triggered-like beats. Single-cell recording revealed enhanced late sodium currents (INa,L) in AF iPSC-aCMs that were absent in a heterologous expression model. Gene expression profiling of AF iPSC-aCMs showed differential expression of the nitric oxide (NO)-mediated signaling pathway underlying enhanced INa,L. We showed that patient-specific AF iPSC-aCMs exhibited striking in vitro electrophysiological phenotype of AF-linked SCN5A mutations, and transcriptomic analyses supported that the NO signaling pathway modulated the INa,L and triggered AF.

Genomics-Driven Activation of Silent Biosynthetic Gene Clusters in Burkholderia gladioli by Screening Recombineering System.

The Burkholderia genus possesses ecological and metabolic diversities. A large number of silent biosynthetic gene clusters (BGCs) in the Burkholderia genome remain uncharacterized and represent a promising resource for new natural product discovery. However, exploitation of the metabolomic potential of Burkholderia is limited by the absence of efficient genetic manipulation tools. Here, we screened a bacteriophage recombinase system Redγ-BAS, which was functional for genome modification in the plant pathogen Burkholderia gladioli ATCC 10248. By using this recombineering tool, the constitutive promoters were precisely inserted in the genome, leading to activation of two silent nonribosomal peptide synthetase gene clusters (bgdd and hgdd) and production of corresponding new classes of lipopeptides, burriogladiodins A-H (1-8) and haereogladiodins A-B (9-10). Structure elucidation revealed an unnatural amino acid Z- dehydrobutyrine (Dhb) in 1-8 and an E-Dhb in 9-10. Notably, compounds 2-4 and 9 feature an unusual threonine tag that is longer than the predicted collinearity assembly lines. The structural diversity of burriogladiodins was derived from the relaxed substrate specificity of the fifth adenylation domain as well as chain termination conducted by water or threonine. The recombinase-mediating genome editing system is not only applicable in B. gladioli, but also possesses great potential for mining meaningful silent gene clusters from other Burkholderia species.

Engineering and elucidation of the lipoinitiation process in nonribosomal peptide biosynthesis.

Nonribosomal peptide synthetases containing starter condensation domains direct the biosynthesis of nonribosomal lipopeptides, which generally exhibit wide bioactivities. The acyl chain has strong impacts on bioactivity and toxicity, but the lack of an in-depth understanding of starter condensation domain-mediated lipoinitiation limits the bioengineering of NRPSs to obtain novel derivatives with desired acyl chains. Here, we show that the acyl chains of the lipopeptides rhizomide, holrhizin, and glidobactin were modified by engineering the starter condensation domain, suggesting a workable approach to change the acyl chain. Based on the structure of the mutated starter condensation domain of rhizomide biosynthetic enzyme RzmA in complex with octanoyl-CoA and related point mutation experiments, we identify a set of residues responsible for the selectivity of substrate acyl chains and extend the acyl chains from acetyl to palmitoyl. Furthermore, we illustrate three possible conformational states of starter condensation domains during the reaction cycle of the lipoinitiation process. Our studies provide further insights into the mechanism of lipoinitiation and the engineering of nonribosomal peptide synthetases.

Development and comprehensive characterization of porcine hepatocellular carcinoma for translational liver cancer investigation.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide. New animal models that faithfully recapitulate human HCC phenotypes are required to address unmet clinical needs and advance standard-of-care therapeutics. This study utilized the Oncopig Cancer Model to develop a translational porcine HCC model which can serve as a bridge between murine studies and human clinical practice. Reliable development of Oncopig HCC cell lines was demonstrated through hepatocyte isolation and Cre recombinase exposure across 15 Oncopigs. Oncopig and human HCC cell lines displayed similar cell cycle lengths, alpha-fetoprotein production, arginase-1 staining, chemosusceptibility, and drug metabolizing enzyme expression. The ability of Oncopig HCC cells to consistently produce tumors in vivo was confirmed via subcutaneous (SQ) injection into immunodeficient mice and Oncopigs. Reproducible development of intrahepatic tumors in an alcohol-induced fibrotic microenvironment was achieved via engraftment of SQ tumors into fibrotic Oncopig livers. Whole-genome sequencing demontrated intrahepatic tumor tissue resembled human HCC at the genomic level. Finally, Oncopig HCC cells are amenable to gene editing for development of personalized HCC tumors. This study provides a novel, clinically-relevant porcine HCC model which holds great promise for improving HCC outcomes through testing of novel therapeutic approaches to accelerate and enhance clinical trials.

Identification of Holrhizins E-Q Reveals the Diversity of Nonribosomal Lipopeptides in Paraburkholderia rhizoxinica.

The products of a nonribosomal peptide synthetase gene, holA, from Paraburkholderia rhizoxinica were investigated using our recently established recombineering technique. Fifteen products, including 13 new linear lipopeptides, holrhizins E-Q (2-8, 10-15), together with the two known holrhizins A and B (1, 9), were detected in the activated mutant, and their structures were identified using HRESIMS, NMR spectroscopy, Marfey's analysis, and feeding experiments with labeled amino acids. The lipohexapeptides 1-3 and 7-14 differ in three amino acid residues and the N-terminal fatty acid chains. The diversity of the holrhizins originates from the substrate flexibility of the A4, A5, and A6 domains as well as the starter C domain in the biosynthetic pathway.

Biosynthesis of polyketides by trans-AT polyketide synthases in Burkholderiales.

Widely used as drugs and agrochemicals, polyketides are a family of bioactive natural products, with diverse structures and functions. Polyketides are produced by megaenzymes termed as polyketide synthases (PKSs). PKS biosynthetic pathways are divided into the cis-AT PKSs and trans-AT PKSs; a division based mainly on the absence of an acyltransferase (AT) domain in the trans-AT PKS modules. In trans-AT biosynthesis, the AT activity is contributed via one or several independent proteins, and there are few other characteristics that distinguish trans-AT PKSs from cis-AT PKSs, especially in the formation of the ß-branch. The trans-AT PKSs constitute a major PKS pathway, and many are found in Burkholderia species, which are prevalent in the environment and prolific sources of polyketides. This review summarizes studies from 1973 to 2017 on the biosynthesis of natural products by trans-AT PKSs from Burkholderia species.

Single-Stranded DNA-Binding Protein and Exogenous RecBCD Inhibitors Enhance Phage-Derived Homologous Recombination in Pseudomonas.

The limited efficiency of the available tools for genetic manipulation of Pseudomonas limits fundamental research and utilization of this genus. We explored the properties of a lambda Red-like operon (BAS) from Pseudomonas aeruginosa phage Ab31 and a Rac bacteriophage RecET-like operon (RecTEPsy) from Pseudomonas syringae pv. syringae B728a. Compared with RecTEPsy, the BAS operon was functional at a higher temperature indicating potential to be a generic system for Pseudomonas. Owing to the lack of RecBCD inhibitor in the BAS operon, we added Redγ or Pluγ and found increased recombineering efficiencies in P. aeruginosa and Pseudomonas fluorescens but not in Pseudomonas putida and P. syringae. Overexpression of single-stranded DNA-binding protein enhanced recombineering in several contexts including RecET recombination in E. coli. The utility of these systems was demonstrated by engineering P. aeruginosa genomes to create an attenuated rhamnolipid producer. Our work enhances the potential for functional genomics in Pseudomonas.

Recombineering Pseudomonas protegens CHA0: An innovative approach that improves nitrogen fixation with impressive bactericidal potency.

Pseudomonas protegens CHA0 is a well-characterized, root-colonizing bacterium with broad-spectrum biocontrol ability. Therefore, it has a great potential to curb plant diseases and to partly replace synthetic chemical pesticides that are harmful to humans. Here, we obtained the multifunctional mutant CHA0-ΔretS-Nif via Red/ET recombineering technology. After deletion of the retS gene and integration of the nitrogen-fixing gene island (Nif) into the CHA0 genome, the resulting mutant, CHA0-ΔretS-Nif, manifested improved both bactericidal activity and biological nitrogen-fixation function. A pot experiment of Arabidopsis thaliana indicated that the strain CHA0-ΔretS-Nif promoted plant growth via expressing several secondary factors, such as the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) and nitrogenase. In order to grow this biocontrol strain at an industrial level, the growth conditions in a 1 L continuous-flow fermenter were optimized to 28 °C, pH of 7.0, and 600 rpm. Moreover, growth experiments in a 5 L fermenter with these optimal growth conditions yielded the maximum cell density, providing vital insights for the industrialization and large-scale fermentation of P. protegens CHA0 for further applications. CHA0-ΔretS-Nif possesses both bactericidal and nitrogen-fixation activities and thus could be used as a biological agent to enhance crop production.