Lysobacter enzymogenes is less-studied, but emerging as a powerful biocontrol bacterium producing multiple antimicrobial weapons including lytic enzymes, toxins, secondary metabolites and protein secretion systems.The...Lysobacter enzymogenes is less-studied, but emerging as a powerful biocontrol bacterium producing multiple antimicrobial weapons including lytic enzymes, toxins, secondary metabolites and protein secretion systems.The loss of surface-attached flagellum, production of heat-stable antifungal factor(HSAF, also named as Ningrongmycin) as a novel antifungal antibiotic, and the use of the type Ⅳ secretion system(T4SS) rather than the common type Ⅵ secretion system(T6SS) to kill competitor bacteria make this species unique. These distinct features set L. enzymogenes apart from well-studied plant beneficial biocontrol agents, such as Bacillus and Pseudomonas. This review describes what takes L. enzymogenes to be a unique biocontrol warrior by focusing to illustrate how the lack of flagellum governs morphological and functional co-adaptability, what adapted signaling transduction pathways are adopted to coordinate the biosynthesis of HSAF, and how to ecologically adapt plant rhizosphere by cell-to-cell interacting with microbiome members via the bacterial-killing T4SS.展开更多
Bacterial species of the genus Lysobacter are environmentally ubiquitous with strong antifungal biocontrol potential.Heat-stable antifungal factor(HSAF)secreted by the biocontrol bacterium Lysobacter enzymogenes OH11 ...Bacterial species of the genus Lysobacter are environmentally ubiquitous with strong antifungal biocontrol potential.Heat-stable antifungal factor(HSAF)secreted by the biocontrol bacterium Lysobacter enzymogenes OH11 has broad-spectrum and highly efficient antifungal activity.Studying the biosynthetic regulations of HSAF would lay an important foundation for strain engineering toward improved HSAF production.In this work,we demonstrate that Le0752,an orotidine-5´-phosphate decarboxylase enzyme(ODCase)catalyzing a pivotal step of the UMP de novo biosynthesis pathway,is vital for HSAF-mediated antimicrobial activities and growth of L.enzymogenes OH11,but not for twitching motility.This gene regulates the production of HSAF by affecting the expression of lafB,a key gene in the HSAF biosynthesis operon,through the transcription factor Clp.Interestingly,bioinformatics analysis revealed that Le0752 belongs to the Group III ODCases,whereas its homologs in the closely related genera Xanthomonas and Stenotrophomonas belong to Group I,which contains most ODCases from Gram-positive bacteria,Gram-negative bacteria and cyanobacteria.Moreover,the Group I ODCase PXO_3614 from the Xanthomonas oryzae pv.oryzae PXO99A strain complemented the Le0752 mutant in regulating HSAF-mediated antagonistic activity.Together,these results highlight the important requirement of de novo pyrimidine biosynthetic enzymes for antibiotic HSAF production in L.enzymogenes,which lays an important foundation for improving HSAF production via metabolic flow design and for dissecting the regulatory functions of bacterial ODCases.展开更多
Strain OH11, a Gram-negative, nonspore forming, rod-shaped bacterium with powerful antagonistic activity, was isolated from rhizosphere of green pepper in Jiangsu Academy of Agricultural Sciences of China and characte...Strain OH11, a Gram-negative, nonspore forming, rod-shaped bacterium with powerful antagonistic activity, was isolated from rhizosphere of green pepper in Jiangsu Academy of Agricultural Sciences of China and characterized to determine its taxonomic position. 16S rRNA gene sequence analysis revealed that strain OH11 belongs to the Gammaproteobacteria and had the highest degree of sequence similarity to Lysobacter enzymogenes strain C3 （AY074793） （99%）, Lysobacter enzymogenes strain N4-7 （U89965） （99%）, Lysobacter antibioticus strain （AB019582） （97%）, and Lysobacter gummosus strain （AB16136） （97%）. Chemotaxonomic data revealed that strain OHI 1 possesses a quinine system with Q-8 as the predominant compound and C15：0 iso, C17：1 iso ω9c as the predominant iso-branched fatty acids, all of which corroborated the assignment of strain OH11 to the genus Lysobacter. Results of DNA-DNA hybridization and physiological and biochemical tests clearly showed that strain OH11 was classified as Lysobacter enzymogenes. Strain OH11 could produce protease, chitinase, and β-1,3-glucanase. It showed strong in vitro antifungal activity against Rhizoctonia solani, Sclerotinia scletotiorum, and several other phytopathogenic fungi. This is the first report of identification and characterization of Lysobacter enzymogenes as a biological control agent of plant diseases in China.展开更多
Lysobacter harbors a plethora of cryptic biosynthetic gene clusters(BGCs),albeit only a limited number have been analyzed to date.In this study,we described the activation of a cryptic polyketide synthase(PKS)/nonribo...Lysobacter harbors a plethora of cryptic biosynthetic gene clusters(BGCs),albeit only a limited number have been analyzed to date.In this study,we described the activation of a cryptic polyketide synthase(PKS)/nonribosomal peptide synthetase(NRPS)gene cluster(lsh)in Lysobacter sp.DSM 3655 through promoter engineering and heterologous expression in Streptomyces sp.S001.As a result of this methodology,we were able to isolate two novel linear lipopeptides,lysohexaenetides A(1)and B(2),from the recombinant strain S001-lsh.Furthermore,we proposed the biosynthetic pathway for lysohexaenetides and identified LshA as another example of entirely iterative bacterial PKSs.This study highlights the potential of heterologous expression systems in uncovering cryptic biosynthetic pathways in Lysobacter genomes,particularly in the absence of genetic manipulation tools.展开更多
An aerobic, Gram-negative bacterium, strain 2-5~T, was isolated from a crude oil-contaminated marine sponge collected near Dalian Bay, China, and subjected to a polyphasic taxonomic investigation. Cells of strain 2-5~...An aerobic, Gram-negative bacterium, strain 2-5~T, was isolated from a crude oil-contaminated marine sponge collected near Dalian Bay, China, and subjected to a polyphasic taxonomic investigation. Cells of strain 2-5~T were non-spore forming, non-motile, rods 0.2–0.3 μm wide and 1.1–1.2μm long. Strain 2-5~T grew well on nutrient agar, ~TSA, R2 A agar and LB agar. Colonies of strain 2-5~T on LB agar were circular, smooth with entire margins, non-transparent and pale yellow after 3 d of incubation at 30℃. Growth of strain 2-5~T occurred in LN medium with 0–6% Na Cl; no growth occurred in the presence of 8.0% NaCl. Strain 2-5~T grew at 15–42℃ and at pH 6.0–8.0. Comparative 16 S r RNA gene sequence analysis showed that strain 2-5~T clustered with the species of the genus Lysobacter. Its closet neighbors were the type strains of Lysobacter concretionis KC^TC 12205~T(97% similarity), Lysobacter arseniciresistens ZS79~T(96%), and Lysobacter defluii APB-9 ~T(96%). ~The value for DNA-DNA relatedness between strain 2-5~~T and L. concretionis KC^TC 12205 ~T was 23%. Branched fatty acids iso-C16: 0, iso-C15: 0, iso-C11: 0 3-OH, iso-C17: 1ω9c and iso-C11: 0 were found to be predominant. ~The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. Strain 2-5~T had a DNA G+C content of 63.8 mol%. On the basis of the phenotypic, chemotaxonomic, DNA-DNA hybridization and phylogenetic data, strain 2-5~T represents a novel species of the genus Lysobacter, for which the name Lysobacter hymeniacidonis sp. nov. is proposed. ~The type strain is 2-5~T(=CGMCC 1.12190 ~T = JCM 18137~T).展开更多
Naturally-occurring environmental microorganisms may provide ‘green’ andeffective biocontrol tools for disease management in agricultural crops. Due tothe constant threat of resistant pathogens there is a pressing a...Naturally-occurring environmental microorganisms may provide ‘green’ andeffective biocontrol tools for disease management in agricultural crops. Due tothe constant threat of resistant pathogens there is a pressing and continualneed to search for new biocontrol tools. This study investigated the productionof new analogs of WAP-8294A compounds by the biocontrol agent Lysobacterenzymogenes OH11 through biosynthetic engineering. WAP-8294As are afamily of natural cyclic lipodepsipeptides with potent activity against Grampositive bacteria. A series of genetic manipulations was therefore conductedon the accessory genes in the WAP biosynthetic gene cluster. The resultingstrains containing a single-point mutation in ORF4, which was predicted toencode a 2-ketoglutarate dependent dioxygenase, produced deoxy-WAP8294As. This result provides evidence for the function of ORF4 in catalyzing βhydroxylation of the D-asparagine residue in WAP-8294As. In addition, six newanalogs of WAP-8294As were identified by UHPLC-HR-MS/MS. This is the firstattempt to produce new WAP-8294As in Lysobacter and shows that thespectrum of the biocontrol compounds may be expanded through themanipulation of biosynthetic genes.展开更多
The potential use ofcomposted wood fibre waste （WFW） for the cultivation of bacterial antagonists of Sclerotinia minor was examined with the result that a mix of millet seed （20% w/w） and WFW, suitably amended wit...The potential use ofcomposted wood fibre waste （WFW） for the cultivation of bacterial antagonists of Sclerotinia minor was examined with the result that a mix of millet seed （20% w/w） and WFW, suitably amended with nutrients, proved to be an ideal matrix for the growth of some of these bacteria. Densities in terms ofcfu＇s ranged from 8.5 IOgl0 cfu/g dw to 10.5 logl0 cfu/g dw ullder sterile conditions after 14 days incubation. Lower population densities of the antagonists were achieved under non-sterile conditions in the compost： millet mix of between 7.9-9.3 logm cfu/g dw at the same period. However, when applied in a pot （glasshouse） trial to protect against S. minor, the millet seed appeared to stimulate the growth of this pathogen resulting in a high incidence of attack of lettuce plants after 2-3 weeks. Although the percentage of healthy seedlings increased following application of compost mix grown antagonists （at a rate of 5% v/v） when compared to the control treatment, these values were not statistically significant （p〉0.05） in most cases. Therefore, the use of millet seeds cannot be recommended as a nutrient supplement for the bacterial antagonist cultivation, if to be subsequently used to control fungal pathogens in the field.展开更多
基金funded by the the Fundamental Research Funds for the Central Universities, China (RENCAI2024002 and KJJQ2024014)the Natural Key Research and Development Program of China (2022YFD1400200)the National Natural Science Foundation of China (U22A20486, 32072470, 32001955 and 32470112)
文摘Lysobacter enzymogenes is less-studied, but emerging as a powerful biocontrol bacterium producing multiple antimicrobial weapons including lytic enzymes, toxins, secondary metabolites and protein secretion systems.The loss of surface-attached flagellum, production of heat-stable antifungal factor(HSAF, also named as Ningrongmycin) as a novel antifungal antibiotic, and the use of the type Ⅳ secretion system(T4SS) rather than the common type Ⅵ secretion system(T6SS) to kill competitor bacteria make this species unique. These distinct features set L. enzymogenes apart from well-studied plant beneficial biocontrol agents, such as Bacillus and Pseudomonas. This review describes what takes L. enzymogenes to be a unique biocontrol warrior by focusing to illustrate how the lack of flagellum governs morphological and functional co-adaptability, what adapted signaling transduction pathways are adopted to coordinate the biosynthesis of HSAF, and how to ecologically adapt plant rhizosphere by cell-to-cell interacting with microbiome members via the bacterial-killing T4SS.
基金supported by the National Natural Science Foundation of China(32102283 to Mingming Yang)the Science and Technology Major Project of China National Tobacco Corporation(110202101056(LS-16))the Science and Technology Project of Shaanxi Branch of China National Tobacco Corporation(KJ-2021-02 and KJ-2022-04).
文摘Bacterial species of the genus Lysobacter are environmentally ubiquitous with strong antifungal biocontrol potential.Heat-stable antifungal factor(HSAF)secreted by the biocontrol bacterium Lysobacter enzymogenes OH11 has broad-spectrum and highly efficient antifungal activity.Studying the biosynthetic regulations of HSAF would lay an important foundation for strain engineering toward improved HSAF production.In this work,we demonstrate that Le0752,an orotidine-5´-phosphate decarboxylase enzyme(ODCase)catalyzing a pivotal step of the UMP de novo biosynthesis pathway,is vital for HSAF-mediated antimicrobial activities and growth of L.enzymogenes OH11,but not for twitching motility.This gene regulates the production of HSAF by affecting the expression of lafB,a key gene in the HSAF biosynthesis operon,through the transcription factor Clp.Interestingly,bioinformatics analysis revealed that Le0752 belongs to the Group III ODCases,whereas its homologs in the closely related genera Xanthomonas and Stenotrophomonas belong to Group I,which contains most ODCases from Gram-positive bacteria,Gram-negative bacteria and cyanobacteria.Moreover,the Group I ODCase PXO_3614 from the Xanthomonas oryzae pv.oryzae PXO99A strain complemented the Le0752 mutant in regulating HSAF-mediated antagonistic activity.Together,these results highlight the important requirement of de novo pyrimidine biosynthetic enzymes for antibiotic HSAF production in L.enzymogenes,which lays an important foundation for improving HSAF production via metabolic flow design and for dissecting the regulatory functions of bacterial ODCases.
基金supported by the National High Technology Research and Development Program of China(2006AA10A211)
文摘Strain OH11, a Gram-negative, nonspore forming, rod-shaped bacterium with powerful antagonistic activity, was isolated from rhizosphere of green pepper in Jiangsu Academy of Agricultural Sciences of China and characterized to determine its taxonomic position. 16S rRNA gene sequence analysis revealed that strain OH11 belongs to the Gammaproteobacteria and had the highest degree of sequence similarity to Lysobacter enzymogenes strain C3 （AY074793） （99%）, Lysobacter enzymogenes strain N4-7 （U89965） （99%）, Lysobacter antibioticus strain （AB019582） （97%）, and Lysobacter gummosus strain （AB16136） （97%）. Chemotaxonomic data revealed that strain OHI 1 possesses a quinine system with Q-8 as the predominant compound and C15：0 iso, C17：1 iso ω9c as the predominant iso-branched fatty acids, all of which corroborated the assignment of strain OH11 to the genus Lysobacter. Results of DNA-DNA hybridization and physiological and biochemical tests clearly showed that strain OH11 was classified as Lysobacter enzymogenes. Strain OH11 could produce protease, chitinase, and β-1,3-glucanase. It showed strong in vitro antifungal activity against Rhizoctonia solani, Sclerotinia scletotiorum, and several other phytopathogenic fungi. This is the first report of identification and characterization of Lysobacter enzymogenes as a biological control agent of plant diseases in China.
基金supported by the National Natural Science Foundation of China(Nos.81573311 and 82173702).
文摘Lysobacter harbors a plethora of cryptic biosynthetic gene clusters(BGCs),albeit only a limited number have been analyzed to date.In this study,we described the activation of a cryptic polyketide synthase(PKS)/nonribosomal peptide synthetase(NRPS)gene cluster(lsh)in Lysobacter sp.DSM 3655 through promoter engineering and heterologous expression in Streptomyces sp.S001.As a result of this methodology,we were able to isolate two novel linear lipopeptides,lysohexaenetides A(1)and B(2),from the recombinant strain S001-lsh.Furthermore,we proposed the biosynthetic pathway for lysohexaenetides and identified LshA as another example of entirely iterative bacterial PKSs.This study highlights the potential of heterologous expression systems in uncovering cryptic biosynthetic pathways in Lysobacter genomes,particularly in the absence of genetic manipulation tools.
基金supported by the National Natural Science Foundation of China(31100092)the Hundred Talent Program of the Chinese Academy of Sciences(No.A1097)the Ningbo Natural Science Foundation of China(2011A610028)
文摘An aerobic, Gram-negative bacterium, strain 2-5~T, was isolated from a crude oil-contaminated marine sponge collected near Dalian Bay, China, and subjected to a polyphasic taxonomic investigation. Cells of strain 2-5~T were non-spore forming, non-motile, rods 0.2–0.3 μm wide and 1.1–1.2μm long. Strain 2-5~T grew well on nutrient agar, ~TSA, R2 A agar and LB agar. Colonies of strain 2-5~T on LB agar were circular, smooth with entire margins, non-transparent and pale yellow after 3 d of incubation at 30℃. Growth of strain 2-5~T occurred in LN medium with 0–6% Na Cl; no growth occurred in the presence of 8.0% NaCl. Strain 2-5~T grew at 15–42℃ and at pH 6.0–8.0. Comparative 16 S r RNA gene sequence analysis showed that strain 2-5~T clustered with the species of the genus Lysobacter. Its closet neighbors were the type strains of Lysobacter concretionis KC^TC 12205~T(97% similarity), Lysobacter arseniciresistens ZS79~T(96%), and Lysobacter defluii APB-9 ~T(96%). ~The value for DNA-DNA relatedness between strain 2-5~~T and L. concretionis KC^TC 12205 ~T was 23%. Branched fatty acids iso-C16: 0, iso-C15: 0, iso-C11: 0 3-OH, iso-C17: 1ω9c and iso-C11: 0 were found to be predominant. ~The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. Strain 2-5~T had a DNA G+C content of 63.8 mol%. On the basis of the phenotypic, chemotaxonomic, DNA-DNA hybridization and phylogenetic data, strain 2-5~T represents a novel species of the genus Lysobacter, for which the name Lysobacter hymeniacidonis sp. nov. is proposed. ~The type strain is 2-5~T(=CGMCC 1.12190 ~T = JCM 18137~T).
文摘Naturally-occurring environmental microorganisms may provide ‘green’ andeffective biocontrol tools for disease management in agricultural crops. Due tothe constant threat of resistant pathogens there is a pressing and continualneed to search for new biocontrol tools. This study investigated the productionof new analogs of WAP-8294A compounds by the biocontrol agent Lysobacterenzymogenes OH11 through biosynthetic engineering. WAP-8294As are afamily of natural cyclic lipodepsipeptides with potent activity against Grampositive bacteria. A series of genetic manipulations was therefore conductedon the accessory genes in the WAP biosynthetic gene cluster. The resultingstrains containing a single-point mutation in ORF4, which was predicted toencode a 2-ketoglutarate dependent dioxygenase, produced deoxy-WAP8294As. This result provides evidence for the function of ORF4 in catalyzing βhydroxylation of the D-asparagine residue in WAP-8294As. In addition, six newanalogs of WAP-8294As were identified by UHPLC-HR-MS/MS. This is the firstattempt to produce new WAP-8294As in Lysobacter and shows that thespectrum of the biocontrol compounds may be expanded through themanipulation of biosynthetic genes.
文摘The potential use ofcomposted wood fibre waste （WFW） for the cultivation of bacterial antagonists of Sclerotinia minor was examined with the result that a mix of millet seed （20% w/w） and WFW, suitably amended with nutrients, proved to be an ideal matrix for the growth of some of these bacteria. Densities in terms ofcfu＇s ranged from 8.5 IOgl0 cfu/g dw to 10.5 logl0 cfu/g dw ullder sterile conditions after 14 days incubation. Lower population densities of the antagonists were achieved under non-sterile conditions in the compost： millet mix of between 7.9-9.3 logm cfu/g dw at the same period. However, when applied in a pot （glasshouse） trial to protect against S. minor, the millet seed appeared to stimulate the growth of this pathogen resulting in a high incidence of attack of lettuce plants after 2-3 weeks. Although the percentage of healthy seedlings increased following application of compost mix grown antagonists （at a rate of 5% v/v） when compared to the control treatment, these values were not statistically significant （p〉0.05） in most cases. Therefore, the use of millet seeds cannot be recommended as a nutrient supplement for the bacterial antagonist cultivation, if to be subsequently used to control fungal pathogens in the field.
