Mechanisms of Pathogen and Pesticide Resistance in Honey Bees.

Bees are the most important insect pollinators of the crops humans grow, and Apis mellifera, the Western honey bee, is the most commonly managed species for this purpose. In addition to providing agricultural services, the complex biology of honey bees has been the subject of scientific study since the 18th century, and the intricate behaviors of honey bees and ants, fellow hymenopterans, inspired much sociobiological inquest. Unfortunately, honey bees are constantly exposed to parasites, pathogens, and xenobiotics, all of which pose threats to their health. Despite our curiosity about and dependence on honey bees, defining the molecular mechanisms underlying their interactions with biotic and abiotic stressors has been challenging. The very aspects of their physiology and behavior that make them so important to agriculture also make them challenging to study, relative to canonical model organisms. However, because we rely on A. mellifera so much for pollination, we must continue our efforts to understand what ails them. Here, we review major advancements in our knowledge of honey bee physiology, focusing on immunity and detoxification, and highlight some challenges that remain.

Genomic diversity in Paenibacillus polymyxa: unveiling distinct species groups and functional variability.

BACKGROUND: Paenibacillus polymyxa is a bacterial species of high interest, as suggested by the increased number of publications on its functions in the past years. Accordingly, the number of described strains and sequenced genomes is also on the rise. While functional diversity of P. polymyxa has been suggested before, the available genomic data is now sufficient for robust comparative genomics analyses. RESULTS: Using 157 genomes, we found significant disparities among strains currently affiliated to P. polymyxa. Multiple taxonomic groups were identified with conserved predicted functions putatively impacting their respective ecology. As strains of this species have been reported to exhibit considerable potential in agriculture, medicine, and bioremediation, it is preferable to clarify their taxonomic organization to facilitate reliable and durable approval as active ingredients. CONCLUSIONS: Strains currently affiliated to P. polymyxa can be separated into two major species groups with differential potential in nitrogen fixation, plant interaction, secondary metabolism, and antimicrobial resistance, as inferred from genomic data.

Analysis of the complete genome sequence of Paenibacillus sp. lzh-N1 reveals its antagonistic ability.

BACKGROUND: Plant diseases caused by pathogenic fungi are devastating. However, commonly used fungicides are harmful to the environment, and some are becoming ineffective due to fungal resistance. Therefore, eco-friendly biological methods to control pathogenic fungi are urgently needed. RESULTS: In this study, a strain, Paenibacillus sp. lzh-N1, that could inhibit the growth of the pathogenic fungus Mycosphaerella sentina (Fr) Schrorter was isolated from the rhizosphere soil of pear trees, and the complete genome sequence of the strain was obtained, annotated, and analyzed to reveal the genetic foundation of its antagonistic ability. The entire genome of this strain contained a circular chromosome of 5,641,488 bp with a GC content of 45.50%. The results of species identification show that the strain belongs to the same species as P. polymyxa Sb3-1 and P. polymyxa CJX518. Sixteen secondary metabolic biosynthetic gene clusters were predicted by antiSMASH, including those of the antifungal peptides fusaricidin B and paenilarvins. In addition, biofilm formation-related genes containing two potential gene clusters for cyclic lactone autoinducer, a gene encoding S-ribosylhomocysteine lyase (LuxS), and three genes encoding exopolysaccharide biosynthesis protein were identified. CONCLUSIONS: Antifungal peptides and glucanase biosynthesized by Paenibacillus sp. lzh-N1 may be responsible for its antagonistic effect. Moreover, quorum sensing systems may influence the biocontrol activity of this strain directly or indirectly.

Metabolic engineering of "last-line antibiotic" colistin in Paenibacillus polymyxa.

Colistin, also known as polymyxin E, is a lipopeptide antibiotic used to treat infections caused by multidrug-resistant gram-negative bacteria. It is considered a "last-line antibiotic", but its clinical development is hindered by low titer and impurities resulting from the presence of diverse homologs in microbial fermentation. To ensure consistent pharmaceutical activity and kinetics, it is crucial to have high-purity colistin active pharmaceutical ingredient (API) in the pharmaceutical industry. This study focused on the metabolic engineering of a natural colistin producer strain to produce colistin with a high titer and purity. Guided by genome mining, we identified Paenibacillus polymyxa ATCC 842 as a natural colistin producer capable of generating a high proportion of colistin A. By systematically inactivating seven non-essential biosynthetic gene clusters (BGCs) of peptide metabolites that might compete precursors with colistin or inhibit colistin production, we created an engineered strain, P14, which exhibited an 82% increase in colistin titer and effectively eliminated metabolite impurities such as tridecaptin, paenibacillin, and paenilan. Additionally, we engineered the L-2,4-diaminobutyric acid (L-2,4-DABA) pathway to further enhance colistin production, resulting in the engineered strain P19, which boosted a remarkable colistin titer of 649.3 mg/L - a 269% improvement compared to the original strain. By concurrently feeding L-isoleucine and L-leucine, we successfully produced high-purity colistin A, constituting 88% of the total colistin products. This study highlights the potential of metabolic engineering in improving the titer and purity of lipopeptide antibiotics in the non-model strain, making them more suitable for clinical use. These findings indicate that efficiently producing colistin API in high purity directly from fermentation can now be achieved in a straightforward manner.

Mining biosynthetic gene clusters in Paenibacillus genomes to discover novel antibiotics.

BACKGROUND: Bacterial antimicrobial resistance poses a severe threat to humanity, necessitating the urgent development of new antibiotics. Recent advances in genome sequencing offer new avenues for antibiotic discovery. Paenibacillus genomes encompass a considerable array of antibiotic biosynthetic gene clusters (BGCs), rendering these species as good candidates for genome-driven novel antibiotic exploration. Nevertheless, BGCs within Paenibacillus genomes have not been extensively studied. RESULTS: We conducted an analysis of 554 Paenibacillus genome sequences, sourced from the National Center for Biotechnology Information database, with a focused investigation involving 89 of these genomes via antiSMASH. Our analysis unearthed a total of 848 BGCs, of which 716 (84.4%) were classified as unknown. From the initial pool of 554 Paenibacillus strains, we selected 26 available in culture collections for an in-depth evaluation. Genomic scrutiny of these selected strains unveiled 255 BGCs, encoding non-ribosomal peptide synthetases, polyketide synthases, and bacteriocins, with 221 (86.7%) classified as unknown. Among these strains, 20 exhibited antimicrobial activity against the gram-positive bacterium Micrococcus luteus, yet only six strains displayed activity against the gram-negative bacterium Escherichia coli. We proceeded to focus on Paenibacillus brasilensis, which featured five new BGCs for further investigation. To facilitate detailed characterization, we constructed a mutant in which a single BGC encoding a novel antibiotic was activated while simultaneously inactivating multiple BGCs using a cytosine base editor (CBE). The novel antibiotic was found to be localized to the cell wall and demonstrated activity against both gram-positive bacteria and fungi. The chemical structure of the new antibiotic was elucidated on the basis of ESIMS, 1D and 2D NMR spectroscopic data. The novel compound, with a molecular weight of 926, was named bracidin. CONCLUSIONS: This study outcome highlights the potential of Paenibacillus species as valuable sources for novel antibiotics. In addition, CBE-mediated dereplication of antibiotics proved to be a rapid and efficient method for characterizing novel antibiotics from Paenibacillus species, suggesting that it will greatly accelerate the genome-based development of new antibiotics.

Efficacy of rhizobacteria Paenibacillus polymyxa SY42 for the biological control of Atractylodes chinensis root rot.

Atractylodes chinensis is one of the most commonly used bulk herbs in East Asia; however, root rot can seriously affect its quality and yields. In contrast to chemical pesticides, biological control strategies are environmentally compatible and safe. For this study, 68 antagonistic bacterial strains were isolated from the rhizospheres of healthy Atractylodes chinensis. Strain SY42 exhibited the most potent fungicidal activities, with inhibition rates against F. oxysporum, F. solani, and F. redolens of 67.07 %, 63.40 % and 68.45 %, respectively. Through morphological observation and molecular characterization, strain SY42 was identified as Paenibacillus polymyxa. The volatile organic components (VOCs) produced by SY42 effectively inhibited the mycelial growth of pathogenic fungi through diffusion. SY42 significantly inhibited the germination of pathogenic fungal spores. Following co-culturing with SY42, the mycelium of the pathogenic fungus was deformed, folded, and even ruptured. SY42 could produce cellulases and proteases to degrade fungal cell walls. Pot experiments demonstrated the excellent biocontrol efficacy of SY42. This study revealed that P. polymyxa SY42 inhibited pathogenic fungi through multiple mechanisms, which verified its utility as a biocontrol agent for the control of A. chinensis root rot.

Microbial cell factories using Paenibacillus: status and perspectives.

Considered a "Generally Recognized As Safe" (GRAS) bacterium, the plant growth-promoting rhizobacterium Paenibacillus has been widely applied in: agriculture, medicine, industry, and environmental remediation. Paenibacillus species not only accelerate plant growth and degrade toxic substances in wastewater and soil but also produce industrially-relevant enzymes and antimicrobial peptides. Due to a lack of genetic manipulation tools and methods, exploitation of the bioresources of naturally isolated Paenibacillus species has long been limited. Genetic manipulation tools and methods continue to improve in Paenibacillus, such as shuttle plasmids, promoters, and genetic tools of CRISPR. Furthermore, genetic transformation systems develop gradually, including: penicillin-mediated transformation, electroporation, and magnesium amino acid-mediated transformation. As genetic manipulation methods of homologous recombination and CRISPR-mediated editing system have developed gradually, Paenibacillus has come to be regarded as a promising microbial chassis for biomanufacturing, expanding its application scope, such as: industrial enzymes, bioremediation and bioadsorption, surfactants, and antibacterial agents. In this review, we describe the applications of Paenibacillus bioproducts, and then discuss recent advances and future challenges in the development of genetic manipulation systems in this genus. This work highlights the potential of Paenibacillus as a new microbial chassis for mining bioresources.

Enzymatic characterization and thermostability improvement of an acidophilic endoxylanase PphXyn11 from Paenibacillus physcomitrellae XB.

GH11 enzyme is known to be specific and efficient for the hydrolysis of xylan. It has been isolated from many microorganisms, and its enzymatic characteristics and thermostability vary between species. In this study, a GH11 enzyme PphXyn11 from a novel xylan-degrading strain of Paenibacillus physcomitrellae XB was characterized, and five mutants were constructed to try to improve the enzyme's thermostability. The results showed that PphXyn11 was an acidophilic endo-ß-1,4-xylanase with the optimal reaction pH of 3.0-4.0, and it could deconstruct different kinds of xylan substrates efficiently, such as beechwood xylan, wheat arabinoxylan and xylo-oligosaccharides, to produce xylobiose and xylotriose as the main products at the optimal reaction temperature of 40 °C. Improvement of the thermal stability of PphXyn11 using site-directed mutagenesis revealed that three mutants, W33C/N47C, S127C/N174C and S49E, designed by adding the disulfide bonds at the N-terminal, C-terminal and increasing the charged residues on the surface of PphXyn11 respectively, could increase the enzymatic activity and thermal stablility significantly and make the optimal reaction temperature reach 50 °C. Molecular dynamics simulations as well as computed the numbers of salt bridges and hydrogen bonds indicated that the protein structures of these three mutants were more stable than the wild type, which provided theoretical support for their improved thermal stability. Certainly, further research is necessary to improve the enzymatic characteristics of PphXyn11 to achieve the bioconversion of hemicellulosic biomass on an applicable scale.

Paenibacillus thermotolerans sp. nov., isolated from a hot spring in Yunnan Province, south-west China.

Two Gram-positive, rod-shaped, endospore-forming strains, YIM B05601 and YIM B05602T, were isolated from soil sampled at Hamazui hot spring, Tengchong City, Yunnan Province, PR China. Phylogenetic analysis based on 16S rRNA gene sequences suggested that the two strains fell within the genus Paenibacillus, appearing most closely related to Paenibacillus alkalitolerans YIM B00362T (96.9 % sequence similarity). Genome-based phylogenetic analysis confirmed that strains YIM B05601 and YIM B05602T formed a distinct phylogenetic cluster within the genus Paenibacillus. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values of strains YIM B05601 and YIM B05602T with the related species P. alkalitolerans YIM B00362T were within the ranges of 74.43-74.57 % and 12.1-18.5 %, respectively, which clearly indicated that strains YIM B05601, YIM B05602T represented a novel species. Strains YIM B05601 and YIM B05602T exhibited 99.6 % 16S rRNA gene sequence similarity. The ANI and dDDH values between the two strains were 99.8 and 100 %, respectively, suggesting that they belong to the same species. Optimum growth for both strains occurred at pH 7.0 and 45 °C. The diagnostic diamino acid in the cell-wall peptidoglycan of strains YIM B05601 and YIM B05602T was meso-diaminopimelic acid. MK-7 was the predominant menaquinone. The polar lipids of strain YIM B05602T were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, four unidentified glycolipids, an unidentified polarlipid and phosphatidylinositol mannoside. The major fatty acids of the two stains were iso-C15 : 0 and anteiso-C15 : 0. Based on phylogenomic and phylogenetic analyses coupled with phenotypic and chemotaxonomic characterizations, strains YIM B05601 and YIM B05602T could be classified as a novel species of the genus Paenibacillus, for which the name Paenibacillus thermotolerans sp. nov. is proposed. The type strain is YIM B05602T (=CGMCC 1.60051T=KCTC 43460T=NBRC 115924T).

Paenibacillus glufosinatiresistens sp. nov., a glufosinate-resistant bacterium isolated from sludge.

A Gram-stain-positive bacterium capable of resisting 5.0 mM glufosinate, designated strain YX-27T, was isolated from a sludge sample collected from a factory in Wuxi, Jiangsu, PR China. Cells were rod-shaped, facultatively anaerobic, endospore-forming, and motile by peritrichous flagella. Growth was observed at 15-42 °C (optimum at 30 °C), pH 4.0-8.0 (optimum pH 7.0-7.5) and with 0-2.5% NaCl (w/v; optimum, 0.5 %). Strain YX-27T could tolerate up to 6.0 mM glufosinate. Strain YX-27T showed the highest 16S rRNA gene sequence similarity to Paenibacillus tianjinensis TB2019T (96.17 %), followed by Paenibacillus odorifer DSM 1539T (96.15 %), Paenibacillus sophorae S27T (96.04 %), Paenibacillus apii 7124T (96.02 %) and Paenibacillus stellifer DSM 14472T (95.87 %). The phylogenetic tree based on genome and 16S rRNA gene sequences indicated that strain YX-27T was clustered in the genus Paenibacillus but formed a separate clade. The genome size of YX-27T was 5.22 Mb with a G+C content of 57.5 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between the genomes of strain YX-27T and 12 closely related type strains ranged from 70.8 to 74.8% and 19.8 to 23.0 %, respectively. The major cellular fatty acids were C16 : 0, anteiso-C15 : 0 and iso-C16 : 0. The major polar lipids were one diphosphatidylglycerol, one phosphatidylethanolamine, one phosphatidylglycerol, one phospholipid, four aminophospholipids and four unidentified lipids. The predominant respiratory quinone was MK-7. Based on phylogenetic, genomic, chemotaxonomic and phenotypic data, strain YX-27T was considered to represent a novel species for which the name Paenibacillus glufosinatiresistens sp. nov. is proposed, with YX-27T (=MCCC 1K08803T= KCTC 43611T) as the type strain.

Paenibacillus hexagrammi sp. nov., a novel bacterium isolated from the gut content of Hexagrammos agrammus.

A Gram-stain-positive, aerobic bacterium, designated as YPD9-1T, was isolated from the gut contents of a spotty belly greenling, Hexagrammos agrammus, collected near Dokdo island, South Korea. The rod-shaped cells were oxidase-positive, and catalase-negative. The major cellular fatty acids were anteiso-C15 : 0, iso-C15 : 0, C16 : 0, iso-C16 : 0 and iso-C17: 0. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and two unidentified lipids. The DNA G+C content was 47.6 mol% and the predominant respiratory quinone was menaquinone MK-7. The 16S rRNA gene sequence of YPD9-1T showed low sequence similarities to species of the genus Paenibacillus, Paenibacillus pocheonensis Gsoil 1138T (97.21 % of sequence similarity), Paenibacillus aestuarii CJ25T (97.12 %) and Paenibacillus allorhizoplanae JJ-42T (96.89 %). The results of phylogenetic analysis based on 16S rRNA gene sequences indicated that YPD9-1T formed a distinct branch among other species of the genus Paenibacillus. The digital DNA-DNA hybridisation, average nucleotide identity, and average amino acid identity values between YPD9-1T and the related species were in the ranges of 15.3-16.2 %, 74.1-78.4 %, and 71.1-71.9 %, respectively, which are below the species cutoff values. On the basis of the results of the polyphasic analysis, we conclude that strain YPD9-1T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus hexagrammi sp. nov. is proposed. The type strain of Paenibacillus hexagrammi is YPD9-1T (=KCTC 43424T =LMG 32988T).

Microbacterium humicola sp. nov., Microbacterium terrisoli sp. nov., Paenibacillus pedocola sp. nov., Paenibacillus silviterrae sp. nov., Flavobacterium terrisoli sp. nov., and Aquabacterium humicola sp. nov., isolated from soil.

Four Gram-stain-positive and two Gram-stain-negative bacterial strains, designated as W4T, FW7T, TW48T, UW52T, PT-3T, and RJY3T, were isolated from soil samples collected from the Republic of Korea. The 16S rRNA gene sequence analysis showed that strains W4T and FW7T belonged to the genus Microbacterium, strains TW48T and UW52T were affiliated to the genus Paenibacillus, strain PT-3T was related to the genus Flavobacterium, and strain RJY3T was associated with the genus Aquabacterium. The closest phylogenetic taxa to W4T, FW7T, TW48T, UW52T, PT-3T, and RJY3T were Microbacterium bovistercoris NEAU-LLET (97.7 %), Microbacterium protaetiae DFW100M-13T (97.9 %), Paenibacillus auburnensis JJ-7T (99.6 %), Paenibacillus allorhizosphaerae JJ-447T (95.7 %), Flavobacterium buctense T7T (97.1 %), and Aquabacterium terrae S2T (99.5 %), respectively. Average nucleotide identity and digital DNA-DNA hybridization values between the novel strains and related reference type strains were <95.0 % and <70.0 %, respectively. The major cellular fatty acid in strains W4T, FW7T TW48T, and UW52T was antiso-C15 : 0. Similarly, strain PT-3T revealed iso-C15 : 0, iso-C15 : 1 G, iso-C17 : 0 3-OH, and iso-C15 : 0 3-OH as its principal fatty acids. On the other hand, RJY3T exhibited summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C16 : 0, summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c), and C12 : 0 as its predominant fatty acids. Overall, the polyphasic taxonomic data indicated that strains W4T, FW7T, TW48T, UW52T, PT-3T, and RJY3T represent novel species within the genera Microbacterium, Paenibacillus, Flavobacterium, and Aquabacterium. Accordingly, we propose the names Microbacterium humicola sp. nov., with the type strain W4T (=KCTC 49888T=NBRC 116001T), Microbacterium terrisoli sp. nov., with the type strain FW7T (=KCTC 49859T=NBRC 116000T), Paenibacillus pedocola sp. nov., with the type strain TW48T (=KCTC 43470T=NBRC 116017T), Paenibacillus silviterrae sp. nov., with the type strain UW52T (=KCTC 43477T=NBRC 116018T), Flavobacterium terrisoli sp. nov., with the type strain PT-3T (=KCTC 92106T=NBRC 116012T), and Aquabacterium humicola sp. nov., with the type strain RJY3T (=KCTC 92105T=NBRC 115831T).

Paenibacillus suaedae sp. nov. and Paenibacillus violae sp. nov., isolated from the roots of Suaeda japonica Makino and Viola mandshurica W. Becker with plant growth-promoting potential.

Two novel Gram-positive strains, chi10T and PFR10T, were isolated from the roots of Suaeda japonica Makino and Viola mandshurica W. Becker, respectively. The strains are facultatively aerobic, rod-shaped, motile via peritrichous flagella and endospore-forming. Strains chi10T and PFR10T showed the highest 16S rRNA gene sequence similarity to Paenibacillus alvei DSM 29T (99.0%) and Paenibacillus planticolens LMG 31457T (99.3%). Furthermore, the phylogenetic and phylogenomic analyses demonstrated that strains chi10T and PFR10T were closely related to the genus Paenibacillus. The digital DNA-DNA hybridization (dDDH) values for strains used in the phylogenetic analysis and strain chi10T ranged from 19.6 to 28.4%, while for strain PFR10T, the values ranged from 19.8 to 53.5%, according to the Genome-to-Genome Distance Calculator 3.0, which were all less than 70%. Additionally, the dDDH value between strains chi10T and PFR10T was 22.0%, confirming that they are different species of the genus Paenibacillus. The average nucleotide identity between chi10T and PFR10T and other species of the genus Paenibacillus were 68.4-84.7% and 67.8-93.6%, respectively. In addition, the major polar lipids of chi10T and PFR10T consisted of diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The major fatty acid profile of strain chi10T was iso-C15 : 0 and anteiso-C15 : 0, whereas the major fatty acid profile of strain PFR10T was iso-C15 : 0, anteiso-C15 : 0 and iso-C16 : 0. The sole respiratory quinone in strains chi10T and PFR10T was menaquinone-7. Phylogenomic, phenotypic and genome analyses strongly indicated that chi10T and PFR10T could be two novel Paenibacillus species for which the names Paenibacillus suaedae sp. nov. (type strain chi10T = KACC 23258T = TBRC 17803T) and Paenibacillus violae sp. nov. (type strain PFR10T = KACC 23263T = TBRC 17804T) are proposed, respectively.

Paenibacillus baimaensis sp. nov., a bacterium isolated from mountain soil in the habitat of Rhinopithecus bieti.

A novel Gram-positive strain WQ 127069T that was isolated from the soil of Baima Snow Mountain, a habitat of highly endangered Yunnan snub-nosed monkeys (Rhinopithecus bieti), was subjected to a polyphasic taxonomic study. Phylogenetic analysis based on the 16S rRNA gene sequences showed that the isolate belongs to the genus Paenibacillus, showing 98.4 and 96.08 % sequence similarity to the type strains Paenibacillus periandrae PM10T and Paenibacillus foliorum LMG 31456T, respectively. The G+C content of the genomic DNA of strain WQ127069T was 45.6 mol%. The predominant isoprenoid quinone was MK-7, and meso-diaminopimelic acid was present in peptidoglycan. The major cellular fatty acids were antiiso-C15 : 0, iso-C15 : 0 and C16 : 0. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and phosphatidylmonomethylethanolamine. The whole genome average nucleotide identity and digital DNA-DNA hybridization values between strain WQ 127069T and strain PM10T were 93.2 and 52.5 %, respectively. Growth occurred at 5-40 °C (optimally at 20-35 °C), pH 6-8 (optimally at pH7.0) and with 0.5-2 % (w/v) NaCl (optimally at 0.5 %). On the basis of the taxonomic evidence, a novel species, Paenibacillus baimaensis sp. nov., is proposed. The type strain is WQ 127069T (=KCTC 43480T=CCTCC AB 2022381T).

Recombinant production of Paenibacillus wynnii ß-galactosidase with Komagataella phaffii.

BACKGROUND: The ß-galactosidase from Paenibacillus wynnii (ß-gal-Pw) is a promising candidate for lactose hydrolysis in milk and dairy products, as it has a higher affinity for the substrate lactose (low KM value) compared to industrially used ß-galactosidases and is not inhibited by the hydrolysis-generated product D-galactose. However, ß-gal-Pw must firstly be produced cost-effectively for any potential industrial application. Accordingly, the yeast Komagataella phaffii was chosen to investigate its feasibility to recombinantly produce ß-gal-Pw since it is approved for the regulated production of food enzymes. The aim of this study was to find the most suitable way to produce the ß-gal-Pw in K. phaffii either extracellularly or intracellularly. RESULTS: Firstly, 11 different signal peptides were tested for extracellular production of ß-gal-Pw by K. phaffii under the control of the constitutive GAP promoter. None of the signal peptides resulted in a secretion of ß-gal-Pw, indicating problems within the secretory pathway of this enzyme. Therefore, intracellular ß-gal-Pw production was investigated using the GAP or methanol-inducible AOX1 promoter. A four-fold higher volumetric ß-galactosidase activity of 7537 ± 66 µkatoNPGal/Lculture was achieved by the K. phaffii clone 27 using the AOX1 promoter in fed-batch bioreactor cultivations, compared to the clone 5 using the GAP promoter. However, a two-fold higher specific productivity of 3.14 ± 0.05 µkatoNPGal/gDCW/h was achieved when using the GAP promoter for ß-gal-Pw production compared to the AOX1 promoter. After partial purification, a ß-gal-Pw enzyme preparation with a total ß-galactosidase activity of 3082 ± 98 µkatoNPGal was obtained from 1 L of recombinant K. phaffii culture (using AOX1 promoter). CONCLUSION: This study showed that the ß-gal-Pw was produced intracellularly by K. phaffii, but the secretion was not achieved with the signal peptides chosen. Nevertheless, a straightforward approach to improve the intracellular ß-gal-Pw production with K. phaffii by using either the GAP or AOX1 promoter in bioreactor cultivations was demonstrated, offering insights into alternative production methods for this enzyme.

Isolation and genome characterization of Paenibacillus polymyxa 188, a potential biocontrol agent against fungi.

AIMS: In this work, we aimed to isolate marine bacteria that produce metabolites with antifungal properties. METHODS AND RESULTS: Paenibacillus polymyxa 188 was isolated from a marine sediment sample, and it showed excellent antifungal activity against many fungi pathogenic to plants (Fusarium tricinctum, Pestalotiopsis clavispora, Fusarium oxysporum, F. oxysporum f. sp. Cubense (Foc), Curvularia plantarum, and Talaromyces pinophilus) and to humans (Aspergillus terreus, Penicillium oxalicum, and Microsphaeropsis arundinis). The antifungal compounds produced by P. polymyxa 188 were extracted and analyzed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The complete genome sequence and biosynthetic gene clusters of P. polymyxa 188 were characterized and compared with those of other strains. A total of 238 carbohydrate-active enzymes (CAZymes) were identified in P. polymyxa 188. Two antibiotic gene clusters, fusaricidin and tridecaptin, exist in P. polymyxa 188, which is different from other strains that typically have multiple antibiotic gene clusters. CONCLUSIONS: Paenibacilluspolymyxa 188 was identified with numerous biosynthetic gene clusters, and its antifungal ability against pathogenic fungi was verified.

Response of Paenibacillus polymyxa SC2 to the stress of polymyxin B and a key ABC transporter YwjA involved.

Polymyxins are cationic peptide antibiotics and regarded as the "final line of defense" against multidrug-resistant bacterial infections. Meanwhile, some polymyxin-resistant strains and the corresponding resistance mechanisms have also been reported. However, the response of the polymyxin-producing strain Paenibacillus polymyxa to polymyxin stress remains unclear. The purpose of this study was to investigate the stress response of gram-positive P. polymyxa SC2 to polymyxin B and to identify functional genes involved in the stress response process. Polymyxin B treatment upregulated the expression of genes related to basal metabolism, transcriptional regulation, transport, and flagella formation and increased intracellular ROS levels, flagellar motility, and biofilm formation in P. polymyxa SC2. Adding magnesium, calcium, and iron alleviated the stress of polymyxin B on P. polymyxa SC2, furthermore, magnesium and calcium could improve the resistance of P. polymyxa SC2 to polymyxin B by promoting biofilm formation. Meanwhile, functional identification of differentially expressed genes indicated that an ABC superfamily transporter YwjA was involved in the stress response to polymyxin B of P. polymyxa SC2. This study provides an important reference for improving the resistance of P. polymyxa to polymyxins and increasing the yield of polymyxins. KEY POINTS: • Phenotypic responses of P. polymyxa to polymyxin B was performed and indicated by RNA-seq • Forming biofilm was a key strategy of P. polymyxa to alleviate polymyxin stress • ABC transporter YwjA was involved in the stress resistance of P. polymyxa to polymyxin B.

Paenibacillus lacisoli sp. nov., a mesotrione-degrading strain isolated from lakeside soil.

A Gram-stain-positive, facultatively anaerobic, rod-shaped bacterium, designated JX-17T, was isolated from a soil sample collected in Jiangxi Province, PR China. Growth was observed at 15-48 °C (optimum 37 °C), at pH 5.0-9.0 (optimum pH 7.0) and with 0-6.0% (w/v) NaCl (optimum 1.0%). Strain JX-17T could degrade approximately 50% of 50 mg/L mesotrione within 2 days of incubation, but could not use mesotrione as sole carbon source for growth. Strain JX-17T showed less than 95.3% 16S rRNA gene sequence similarity with type strains of the genus Paenibacillus. In the phylogenetic tree based on 16S rRNA gene and genome sequences, strain JX-17T formed a distinct lineage within the genus Paenibacillus. The ANI values between JX-17T and the most closely related type strains P. lentus CMG1240T and P. farraposensis UY79T were 70.1% and 71.4%, respectively, and the dDDH values between them were 19.0% and 23.3%, respectively. The major cellular fatty acids were anteiso-C15:0, iso-C16:0, anteiso-C17:0 and C16:0, the predominant respiratory quinone was MK-7, the major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, an unidentified glycolipid, an aminophospholipid and a phosphatidylinositol. The diagnostic diamino acid of the peptidoglycan was meso-diaminopimelic acid, and the DNA G + C content was 50.1 mol%. Based on the phylogenetic, phenotypic and chemotaxonomic characteristics, strain JX-17T represents a novel species within the genus Paenibacillus, for which the name Paenibacillus lacisoli sp. nov is proposed, with strain JX-17T (= GDMCC 1.3962T = KCTC 43568T) as the type strain.

Separation and purification of antimicrobial substances from Paenibacillus polymyxa KH-19 and analysis of its physicochemical characterization.

Soft rot is one of the top ten most dangerous plant pathogens in agricultural production, storage, and transport, and the use of microorganisms and their metabolites to control soft rot is a current research hotspot. In this study, we identified the antimicrobial substance in the metabolite of Paenibacillus polymyxa KH-19, and determined that the antimicrobial substance of this strain was an active protein. The protein was completely precipitated at 40-60% ammonium sulphate saturation and showed good inhibitory effects against seven pathogenic bacteria including Pectobacterium carotovorum BC2 and seven pathogenic fungi including Pyricularia oryzae. The MIC of the protein was 51.563 µg/mL, temperature acid-base UV and light stability insensitive to protease, with high-temperature resistance. The antimicrobial protein was isolated and purified by DEAE-anion exchange column and Sephadex G-75 gel filtration chromatography, and the LC-MS/MS assay identified the protein as lysophosphatidyl esterase with a molecular weight of 25.255 kDa. The purified antimicrobial protein increased the inhibitory effect against P. carotovorum BC2, with the diameter of the circle of inhibition being 26.50 ± 0.915 mm. Bioinformatics analysis showed that the protein has the molecular formula of C1117H1732N316O338S5, encodes 224 amino acids, has an aliphatic index of 88.39, and belongs to the category of hydrophilic unstable proteins. The present study is the first report of an active protein with extreme thermoplastic and resistance to P. carotovorum BC2, which provides a reference for the preparation and application of the antimicrobial substances of P. polymyxa KH-19, as well as a theoretical basis for the study of the function of lysophosphodiesterase protein and its use as a microbial preparation.

Biocontrol Efficacy and Induced Resistance of Paenibacillus polymyxa J2-4 Against Meloidogyne incognita Infection in Cucumber.

Meloidogyne incognita is one of the most destructive agricultural pathogens around the world, resulting in severe damage to yield and quality in agricultural production. Biological control promises to be a great potential alternative to chemical agents against M. incognita. Paenibacillus polymyxa J2-4, isolated from ginger plants injured by M. incognita, has shown excellent biocontrol efficacy against M. incognita in cucumber. In vitro experiments with the strain J2-4 resulted in a correct mortality rate of 88.79% (24 h) and 98.57% (48 h) for second-stage juveniles (J2s) of M. incognita. Strain J2-4 significantly suppressed nematode infection on potted plants, with a 65.94% reduction in galls and a 51.64% reduction in eggs compared with the control. The split-root assay demonstrated that strain J2-4 not only reduced J2s' invasion but also inhibited nematode development through the dependence on salicylic acid and jasmonic acid signaling of strain J2-4 induction of plant resistance in local and systemic roots of cucumbers. Genomic analysis of strain J2-4 indicated biosynthetic gene clusters encoding polymyxin, fusaricidin B, paenilan, and tridecaptin. In addition, genetic analysis showed that none of the genes encoding virulence factors were detected in the genome of J2-4 compared with the pathogenic Bacillus species. Taking all the data together, we conclude that P. polymyxa J2-4 has potential as a biological control agent against M. incognita on cucumbers and can be considered biologically safe when used in agriculture.