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The aim of our study was to upscale the production of Lysobacter enzymogenes strain LEC from shake flasks to a laboratory bioreactor. The obtained fermentation broth was to be downstream-processed by lyophilisation and fluidised bed-drying, followed by testing of the dried preparations against plant pathogens. Different batches of broth from shake flasks and from individual fermenter runs were characterised and compared. Number of viable cells, in vitro efficacy (ED90 value) against the apple scab pathogen Ventura inaequalis, as well as efficacy against Phytophthora infestans on potato plants were all reproducible and not impaired by upscaling the production volume from shake flask to the bioreactor. Fermentation broth was processed by lyophilisation and fluidised bed-drying. In bioassays, the activity against downy mildew (Pseudoperonospora cubensis) on cucumber were comparable for the unformulated fermentation broth and the fluidised bed-dried product, while the lyophilised product was significantly more effective. Strain LEC showed reproducible pathogen suppressive activity, independent from production scale and batch. Production upscaling and formulation by drying are possible without loss of efficacy. [ABSTRACT FROM AUTHOR]

Siderophores are produced by bacteria in iron-restricted conditions. However, we found maltose could induce the biosynthesis of the siderophore lysochelin in Lysobacter sp. 3655 in rich media that are not compatible with siderophore production. Maltose markedly promoted cell growth, with over 300% increase in cell density (OD600) when LB medium was added with maltose (LBM). While lysochelin was not detectable when OD600 in LBM was below 5.0, the siderophore was clearly produced when OD600 reached 7.5 and dramatically increased when OD600 was 15.0. Coincidently, the transcription of lysochelin biosynthesis genes was remarkably enhanced following the increase of OD600. Conversely, the iron concentration in the cell culture dropped to 1.2µM when OD600 reached 15.0, which was 6-fold lower than that in the starting medium. Moreover, mutants of the maltose-utilizing genes (orf2677 and orf2678) or quorum-sensing related gene orf644 significantly lowered the lysochelin yield. Transcriptomics analysis showed that the iron-utilizing/up-taking genes were up-regulated under high cell density. Accordingly, the transcription of lysochelin biosynthetic genes and the yield of lysochelin were stimulated when the iron-utilizing/up-taking genes were deleted. Finally, lysochelin biosynthesis was positively regulated by a TetR regulator (ORF3043). The lysochelin yield in orf3043 mutant decreased to 50% of that in the wild type and then restored in the complementary strain. Together, this study revealed a previously unrecognized mechanism for lysochelin biosynthetic regulation, by which the siderophore could still be massively produced in Lysobacter even grown in a rich culture medium. This finding could find new applications in large-scale production of siderophores in bacteria. [ABSTRACT FROM AUTHOR]

This work investigated the antimicrobial potential of Lysobacter gummosus 10.1.1. The culture fluid of the strain was found to contain antimicrobial agents active against Staphylococcus aureus, Micrococcus luteus, and Bacillus cereus. L. gummosus was first shown to be capable of forming outer membrane vesicles, which have a bacteriolytic effect against not only Gram-positive bacteria but also against the Gram-negative pathogen Pseudomonas aeruginosa. Transcriptomic analysis revealed the genes of almost all known bacteriolytic enzymes of Lysobacter, as well as the genes of enzymes with putative bacteriolytic activity. Also identified were genes involved in the biosynthesis of a number of secondary metabolites for which antimicrobial activities are known. This research is indicative of the relevance of isolating and studying L. gummosus antimicrobial agents. [ABSTRACT FROM AUTHOR]

Microbial secondary metabolites are an important source of natural drugs and are widely used in medical and agricultural production. Actinomycetes and fungi are the main microorganisms that produce antibiotics, but it is becoming increasingly difficult to discover new antibiotics from them after a long and extensive screening. With advances in sequencing technology, more and more microbial genomes are being sequenced, and it has been found that many previously neglected microbes also have the potential to produce new antibiotics. The genus Lysobacter have antagonistic activities against a range of plant pathogenic fungi, bacteria, oomycetes, and nematodes, and several secondary metabolites with novel structures and significant biological activities have been identified from Lysobacter, which have potential applications in biocontrol agents and promising drug leads. In this paper, we review the distribution of Lysobacter resources and their secondary metabolites, aiming to provide a reference for further screening of Lysobacter from the environment and discovering more bioactive natural products. [ABSTRACT FROM AUTHOR]

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Microbial exopolysaccharides (EPSs) have attracted attention from several fields due to their high industrial applicability. In the present study, rhizosphere strain CJ11T was isolated from the root of Glycine max L. in Goyang-si, Republic of Korea, and a novel exopolysaccharide was purified from the Lysobacter sp. CJ11T fermentation broth. The exopolysaccharide's average molecular weight was 0.93 × 105 Da. Its monosaccharide composition included 72.2% mannose, 17.2% glucose, 7.8% galactose, and 2.8% arabinose. Fourier-transform infrared spectroscopy identified the exopolysaccharide carbohydrate polymer functional groups, and the structural properties were investigated using nuclear magnetic resonance. In addition, a microstructure of lyophilized EPS was determined by scanning electron microscopy. Using thermogravimetric analysis, the degradation of the exopolysaccharide produced by strain CJ11T was determined to be 210 °C. The exopolysaccharide at a concentration of 4 mg/mL exhibited 2,2-diphenyl-1-picrylhydrazyl free-radical-scavenging activity of 73.47%. Phylogenetic analysis based on the 16S rRNA gene sequencing results revealed that strain CJ11T was a novel isolate for which the name Lysobacter soyae sp. nov is proposed. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A gram-stain-negative, aerobic, rod-shaped and motile bacterium, designated Alg18-2.2T, was isolated from a bottom sediment of the highly alkaline-saline lake in Buryatia, Russia. The isolate is facultatively alkaliphilic, growing at pH 7.0–12.0 (optimum at 9.0–9.5), and salt-tolerant up to 7% NaCl (w/v; optimum, 1%, w/v). Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain Alg18-2.2T formed a distinct monophyletic lineage within the family Lysobacteraceae. Strain Alg18-2.2T had highest sequence similarity to Lysobacter concretionis Ko07T (95.0%), Lysobacter maris KMU-14T (95.0%), Coralloluteibacterium stylophorae Sty a-1T (94.9%) and Lysobacter psychrotolerans ZS60T (94.8%). The ANI values between Alg18-2.2T and representatives of genera within order Lysobacterales were 66.4–76.5%, which is below of the statistical threshold for the delineation of the species. The AAI values between the strain and representatives of genera within order Lysobacterales were 52.2–66.4%, which is consistent with the data intra-order AAI values for Lysobacterales. The quinone system consisted of ubiquinone 8 (Q-8). Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, two unidentified phospholipids were presented as major lipids in the polar lipid profile. The major cellular fatty acids were 9-Methyl C16:0 (23.9%), iso-C15:0 (22.4%), iso-C16:0 (16.3%) and iso-C17:0 (14.6%). The genome has a G+C content of 68.7 mol % and a total assembly size of 3,025,752 bases. Based on polyphasic results including phylogenomic data, we propose a new genus and species Alkalisalibacterium limincola gen. nov., sp. nov. with the type strain Alg18-2.2T (=CPCC 101300Т = DSM 110880T = VKM B-3394T). [ABSTRACT FROM AUTHOR]

Although synthetic pesticides play a major role in plant protection, their application needs to be reduced because of their negative impact on the environment. This applies also to copper preparations, which are used in organic farming. For this reason, alternatives with less impact on the environment are urgently needed. In this context, we evaluated eight isolates of the genus Lysobacter (mainly Lysobacter enzymogenes) for their activity against plant pathogens. In vitro, the investigated Lysobacter isolates showed broad antagonistic activity against several phytopathogenic fungi, oomycetes and bacteria. Enzyme assays revealed diverse activities for the tested isolates. The most promising L. enzymogenes isolate (LEC) was used for further detailed analyses of its efficacy and effective working concentrations. The experiments included in vitro spore and sporangia germination tests and leaf disc assays as well as ad planta growth chamber trials against Alternaria solani and Phytophthora infestans on tomato plants, Pseudoperonospora cubensis on cucumbers and Venturia inaequalis on young potted apple trees. When applied on leaves, dilutions of a culture suspension of LEC had a concentration-dependent, protective effect against the tested pathogens. In all pathosystems tested, the effective concentrations were in the range of 2.5–5% and similarly efficacious to common plant protection agents containing copper hydroxide, wettable sulphur or fenhexamid. Thus, the isolate of L. enzymogenes identified in this study exhibits a broad activity against common plant pathogens and is therefore a promising candidate for the development of a microbial biocontrol agent. [ABSTRACT FROM AUTHOR]

This work investigated the antimicrobial potential of Lysobacter gummosus 10.1.1. The culture fluid of the strain was found to contain antimicrobial agents active against Staphylococcus aureus, Micrococcus luteus, and Bacillus cereus. L. gummosus was first shown to be capable of forming outer membrane vesicles, which have a bacteriolytic effect against not only Gram-positive bacteria but also against the Gram-negative pathogen Pseudomonas aeruginosa. Transcriptomic analysis revealed the genes of almost all known bacteriolytic enzymes of Lysobacter, as well as the genes of enzymes with putative bacteriolytic activity. Also identified were genes involved in the biosynthesis of a number of secondary metabolites for which antimicrobial activities are known. This research is indicative of the relevance of isolating and studying L. gummosus antimicrobial agents.

● Lysobacter enzymogenes mutants were generated for WAP-8294A biosynthesis. ● Essential and non-essential accessory genes for WAP-8294A biosynthesis were determined. ● Six new WAP-8294A analogs were identified using UHPLC-HR-MS/MS. ● Three deoxy analogs were detected supporting the function of ORF4 in asparagine hydroxylation. Naturally-occurring environmental microorganisms may provide ‘green’ and effective biocontrol tools for disease management in agricultural crops. Due to the constant threat of resistant pathogens there is a pressing and continual need to search for new biocontrol tools. This study investigated the production of new analogs of WAP-8294A compounds by the biocontrol agent Lysobacter enzymogenes OH11 through biosynthetic engineering. WAP-8294As are a family of natural cyclic lipodepsipeptides with potent activity against Gram-positive bacteria. A series of genetic manipulations was therefore conducted on the accessory genes in the WAP biosynthetic gene cluster. The resulting strains containing a single-point mutation in ORF4, which was predicted to encode a 2-ketoglutarate dependent dioxygenase, produced deoxy-WAP-8294As. This result provides evidence for the function of ORF4 in catalyzing β-hydroxylation of the D-asparagine residue in WAP-8294As. In addition, six new analogs of WAP-8294As were identified by UHPLC-HR-MS/MS. This is the first attempt to produce new WAP-8294As in Lysobacter and shows that the spectrum of the biocontrol compounds may be expanded through the manipulation of biosynthetic genes.

ABSTRACT Quorum sensing (QS) is a well-known chemical signaling system responsible for intercellular communication that is widespread in bacteria. Acyl-homoserine lactone (AHL) is the most-studied QS signal. Previously, bacterially encoded AHL-degrading enzymes were considered to be canonical quorum-quenching proteins that have been widely used to control pathogenic infections. Here, we report a novel platform that enabled the efficient discovery of noncanonical AHL quorum-quenching proteins. This platform initially asked bacteriologists to carry out comparative genomic analyses between phylogenetically related AHL-producing and non-AHL-producing members to identify genes that are conservatively shared by non-AHL-producing members but absent in AHL-producing species. These candidate genes were then introduced into recombinant AHL-producing E. coli to screen for target proteins with the ability to block AHL production. Via this platform, we found that non-AHL-producing Lysobacter containing numerous environmentally ubiquitous members encoded a conserved glycosyltransferase-like protein Le4759, which was experimentally shown to be a noncanonical AHL-quenching protein. Le4759 could not directly degrade exogenous AHL but rather recognized and altered the activities of multiple AHL synthases through protein-protein interactions. This versatile capability enabled Le4759 to block specific AHL synthase such as CarI from Pectobacterium carotovorum to reduce its protein abundance to suppress AHL synthesis, thereby impairing bacterial infection. Thus, this study provided bacteriologists with a unique platform to discover noncanonical quorum-quenching proteins that could be developed as promising next-generation drug candidates to overcome emerging bacterial antibiotic resistance. IMPORTANCE Targeting and blocking bacterial quorum sensing (QS), the process known as quorum quenching (QQ) is an effective mean to control bacterial infection and overcome the emerging antibiotic resistance. Previously, diverse QS signal-degradation enzymes are identified as canonical QQ proteins. Here, we provided a novel and universal platform that enabled to discover previously unidentified noncanonical QQ proteins that were unable to degrade acyl-homoserine lactone (AHL) but could block AHL generation by recognizing multiple AHL synthases via direct protein-protein interactions. Our findings are believed to trigger broad interest for bacteriologists to identify potentially widely distributed noncanonical QQ proteins that have great potential for developing next-generation anti-infectious drugs.

The Phytophthora pathogen causes enormous damage to important agricultural plants. This group of filamentous pathogens is phylogenetically distant from fungi, making them difficult to control by most chemical fungicides. Lysobacter enzymogenes OH11 (OH11) is a biocontrol bacterium that secretes HSAF (Heat-Stable Antifungal Factor) as a broad-spectrum antifungal weapon. Here, we showed that OH11 could also control a variety of plant Phytophthora diseases caused by three major oomycetes (P. sojae, P. capsici and P. infestans). We provided abundant evidence to prove that OH11 protected host plants from Phytophthora pathogen infection by inhibiting mycelial growth, digesting cysts, suppressing cyst germination, and eliciting plant immune responses. Interestingly, the former two processes required the presence of HSAF, while the latter two did not. This suggested that L. enzymogenes could prevent Phytophthora infection via multiple previously unknown mechanisms. Therefore, this study showed that L. enzymogenes could serve as a promising alternative resource for promoting plant resistance to multiple Phytophthora pathogens. [ABSTRACT FROM AUTHOR]

The GH19 chitinase Chi19MK from Lysobacter sp. MK9-1 inhibits fungal growth. In this study, the thermal stability of Chi19MK was investigated in buffers of different pH. Chi19MK retained approximately 100 % of its activity at 60 °C for 2 h in a sodium acetate buffer at pH 4.0. However, the enzyme retained approximately 15 % of its activity at 60 °C in potassium phosphate buffer (pH 6.0) and acetate (pH 5.0). Chi19MK was treated with acetate buffer (pH 4.0) for 1-5 h at a temperature range of 60°C-100 °C, Chi19MK retained more than 70 % of its activity after heat treatment at 60 °C, 70 °C, and 80 °C for 5 h, and its activity was reduced to approximately 65 % after treatment at 90 °C for 2 h and at 100 °C for 1 h. The circular dichroism (CD) spectrum of Chi19MK in 10 mM acetate buffer (pH 4.0) was recorded at 80 °C and the CD spectrum showed the collapse of secondary structures. After decreasing the solution temperature from 80 °C to 25 °C, the spectrum was almost restored to the initial state. In contrast, the secondary structures of Chi19MK were not restored in acetate buffer (pH 5.0) or phosphate buffer (pH 6.0) by cooling after heat treatment at 80 °C. In an antifungal assay against Trichoderma reesei, Chi19MK retained its growth inhibition activity after heat treatment at 70 °C in acetate buffer at pH 4.0. These findings suggest that Chi19MK refolds in acetate buffer at pH 4.0, even when its conformation is disrupted by exposure to high-temperature conditions., (Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Three Gram-stain-negative, aerobic, short rod-shaped and motile strains (FXH3W T , SHGZ20W and SMYT11W T ) were isolated from freshwater environments in China. Comparisons based on the 16S rRNA gene sequences indicated that strains FXH3W T and SHGZ20W showed the highest 16S rRNA gene sequence similarity of about 99.6% to ' Luteimonas cellulosilyticus ' MIC 1.5, and strain SMYT11W T showed the highest 16S rRNA gene sequence similarity of 99.8% to Luteimonas fraxinea D4P002 T , respectively. Observing the phylogenetic trees reconstructed based on the 16S rRNA gene sequences, the species of genera Luteimonas and Lysobacter were not monophyletic and often mixed together. The further reconstructed phylogenomic tree and Genome Taxonomy Database also showed that the species of both genera were polyphyletic, implying that the current taxonomic status of the species of both genera was questionable. The calculated OrthoANIu, digital DNA-DNA hybridization and average amino acid sequence identity (AAI) values supported that strains FXH3W T and SHGZ20W should belong to the same novel species and strain SMYT11W T should also represent an independent novel species. Combining the AAI values and phylogenomic analyses, the species of genera Luteimonas and Lysobacter should be reassigned to 12 genera ( Luteimonas , Lysobacter , Cognatiluteimonas , Noviluteimonas , Pseudoluteimonas , Solilutibacter , Agrilutibacter , Cognatilysobacter , Marilutibacter , Novilysobacter , Montanilutibacter and Aerolutibacter ). The AAI values 69.5-76.0% were also proposed as the Lysobacteraceae -specific thresholds for genus delineation. Strain SMYT11W T should represent a novel species of the genus Luteimonas , for which the name Luteimonas flava sp. nov. (type strains SMYT11W T =GDMCC 1.4275 T =KCTC 8304 T ) is proposed. Strains FXH3W T and SHGZ20W should represent a novel species of a new genus Aquilutibacter rugosus gen. nov., sp. nov. The type strain of the type species is FXH3W T (=GDMCC 1.4096 T =KCTC 8154 T ).

The studies of cave bacterial communities worldwide have revealed their potential to produce antibiotic molecules. In Costa Rica, ~400 caves have been identified; however, their microbial diversity and biotechnological potential remain unexplored. In this work, we studied the chemical composition and microbial diversity of a Costa Rican cave (known as the Amblipigida cave) located in Puntarenas, Costa Rica. Additionally, through culture-dependent methods, we evaluated the potential of its microbiota to produce antibiotic molecules. Mineralogical and elemental analyses revealed that the Amblipigida cave is primarily composed of calcite. However, small variations in chemical composition were observed as a result of specific conditions, such as light flashes or the input of organic matter. The 16S rRNA gene metabarcoding revealed an extraordinarily high microbial diversity (with an average Shannon index of ~6.5), primarily comprising bacteria from the phyla Pseudomonadota, Actinomycetota, Firmicutes and Acidobacteriota, with the family Pseudomonadaceae being the most abundant. A total of 93 bacteria were isolated, of which 15% exhibited antibiotic activity against at least one Gram-positive or yeast strain and were classified within the genera Lysobacter , Streptomyces , Pseudomonas , Brevundimonas and Bacillus . These findings underscore the highly diverse nature of cave microbiota and their significant biotechnological potential, particularly in the production of antibiotic compounds.

Resistance to spectinomycin emerged after widely used for treatment of gonorrhea. Previous studies revealed that Lysobacter enzymogenes strain C3 (LeC3) exhibited elevated level of intrinsic resistance to spectinomycin. In this study, we screened a Tn5 transposon mutant library of LeC3 to elucidate the underlying molecular mechanisms of spectinomycin resistance. Insertion sites in 15 out of 19 mutants recovered with decreased spectinomycin resistance were located on two ribosomal RNA operons at different loci, indicating the pivotal role of ribosomal RNAs in conferring spectinomycin resistance in L. enzymogenes. The other mutants harbored mutations in the tuf, rpoD, mltB, and purB genes. Among them, the tuf and rpoD genes, respectively, encode a translation elongation factor Tu and an RNA polymerase primary sigma factor. They both contribute to protein biosynthesis, where ribosomal RNAs play essential roles. The mltB gene, whose product is involved in cell-wall recycling, was not only associated with resistance against spectinomycin, but also conferred resistance to osmotic stress and ampicillin. In addition, mutation of the purB gene, for which its product is involved in the biosynthesis of inosine and adenosine monophosphates, led to decreased spectinomycin resistance. Addition of exogenous adenine at lower concentration in medium restored the growth deficiency in the purB mutant and increased bacterial resistance to spectinomycin. These results suggest that while cell-wall recycling and purine biosynthesis might contribute to spectinomycin resistance, target rRNAs play critical role in spectinomycin resistance in L. enzymogenes. [ABSTRACT FROM AUTHOR]

Microbial exopolysaccharides (EPSs) have attracted attention from several fields due to their high industrial applicability. In the present study, rhizosphere strain CJ11T was isolated from the root of Glycine max L. in Goyang-si, Republic of Korea, and a novel exopolysaccharide was purified from the Lysobacter sp. CJ11T fermentation broth. The exopolysaccharide’s average molecular weight was 0.93 × 105 Da. Its monosaccharide composition included 72.2% mannose, 17.2% glucose, 7.8% galactose, and 2.8% arabinose. Fourier-transform infrared spectroscopy identified the exopolysaccharide carbohydrate polymer functional groups, and the structural properties were investigated using nuclear magnetic resonance. In addition, a microstructure of lyophilized EPS was determined by scanning electron microscopy. Using thermogravimetric analysis, the degradation of the exopolysaccharide produced by strain CJ11T was determined to be 210 °C. The exopolysaccharide at a concentration of 4 mg/mL exhibited 2,2-diphenyl-1-picrylhydrazyl free-radical-scavenging activity of 73.47%. Phylogenetic analysis based on the 16S rRNA gene sequencing results revealed that strain CJ11T was a novel isolate for which the name Lysobacter soyae sp. nov is proposed.

In the present study, exopolysaccharide (EPS) produced by Lysobacter sp. MMG2 (lyEPS) was characterized and purified. The lyEPS-producing strain Lysobacter sp. MMG2 was isolated from the roots of Tagetes patula. When lyEPS was produced in tryptic soy broth with 1% glucose and the lyophilized powder was measured, the yield was found to be 0.67 g/L. The molecular weight (Mw) of lyEPS was 1.01 × 105 Da. Its monosaccharide composition includes 84.24% mannose, 9.73% glucose, 2.55% galactose, 2.77% arabinose, 0.32% xylose, and 0.03% rhamnose. Scanning electron microscopy (SEM) revealed that lyEPS has various round and rough surfaces. Fourier-transform infrared (FTIR) analysis identified its carbohydrate polymer functional groups. Moreover, thermogravimetric analysis of lyEPS revealed two events of mass loss: the first was water loss, which resulted in 3.97% mass loss and the second event occurred at approximately 212 °C. lyEPS could inhibit biofilm-producing pathogenic bacteria without any antimicrobial activity. Furthermore, lyEPS at a concentration of 4 mg/mL could exhibit potent 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical-scavenging activity (89.25%). These results indicate that lyEPS could be a promising candidate for industrial development if its biological activity is further explored. [ABSTRACT FROM AUTHOR]

The GH-16 type β-1,3-glucanase (BgluC16MK) gene of Lysobacter sp. MK9-1 was cloned to study its antifungal activities. BgluC16MK displays amino acid sequence similarity with GluC from L. enzymogenes strain N4-7. BgluC16MK includes a signal sequence, a catalytic domain and carbohydratebinding module family 6-type β-glucan binding domain (B-GBD). The expression of the BgluC16MK gene in Escherichia coli without the signal sequence resulted in antifungal activity at a dose of 0.6-0.8 nmol/disk. However, BgluC16MK displayed antifungal activity at a dose of 0.025 nmol/disk in combination with Chi19MK. Substrate-specific assay revealed that purified BgluC16MK hydrolyzed insoluble curdlan more readily than the soluble substrate. Furthermore, to explore the binding selectivity of B-GBD of BgluC-16MK, we constructed a fusion protein (B-GBD-GFP) using the B-GBD and green fluorescent protein. The activity of the fusion protein against various substrates indicates that B-GBD was selective for glucans with β-1,3-linkages. An additional study demonstrated the binding ability of B-GBD-GFP to the cell-wall of living fungi, such as T. reesei and Aspergillus oryzae. These findings suggest that BgluC16MK can be utilized to generate antifungal enzyme preparations and that the fusion protein B-GBD-GFP can be used to identify the fungal cell surface structure using β-glucans. [ABSTRACT FROM AUTHOR]

A successful homologous expression system based on Lysobacter capsici VKM B-2533T and the plasmid pBBR1-MCS5 was first developed for a promising bacteriolytic enzyme of this bacterium, β-lytic protease (Blp). In the expression strains, blp gene expression under the regulation of the GroEL(A) and T5 promoters increased by 247- and 667-fold, respectively, as compared with the wild-type strain. After the cultivation of the expression strains L. capsici PGroEL(A)-blp and L. capsici PT5-blp, the Blp yield increased by 6.7- and 8.5-fold, respectively, with respect to the wild-type strain. The cultivation of the expression strain L. capsici PT5-blp was successfully scaled up. Under fermentation conditions the yield of the enzyme increased by 1.6-fold. The developed homologous system was used to express the gene of the bacteriolytic serine protease (Serp) of L. capsici VKM B-2533T. The expression of the serp gene in L. capsici PT5-serp increased by 585-fold. The developed homologous system for the gene expression of bacteriolytic Lysobacter enzymes is potentially biotechnologically valuable, and is promising for creating highly efficient expression strains. [ABSTRACT FROM AUTHOR]

Two Gram-stain-negative, mesophilic, strictly aerobic, nonspore forming, and yellow-pigmented strains with rod-shaped cells, designated H21R20T and H23M41T, were isolated from the faeces of an Oriental stork (Ciconia boyciana). Based on 16S rRNA gene sequences, both strains showed the highest similarity (98.3–98.4%) to the type strain of Lysobacter concretionis. Phylogenetic analysis based on the 16S rRNA genes and 92 bacterial core genes showed that strains H21R20T and H23M41T were robustly clustered with L. concretionis Ko07T. Whole genome sequencing revealed that the genomes of both strains were approximately 2.9 Mb in size. The DNA G + C contents of the H21R20T and H23M41T strains were 67.3 and 66.6%, respectively. The two strains showed 80.1–81.7% average nucleotide identity with L. concretionis Ko07T. Strain H21R20T grew optimally at 30°C and pH 8.0 and in the presence of 0.5–3% (wt/vol) NaCl, while strain H23M41T grew optimally at 30°C and pH 7.0–8.0 and in the presence of 0–3% (wt/vol) NaCl. Both strains possessed iso-C15:0, iso-C16:0 and summed feature 9 (iso-C17:1ω9c and/or C16:010-methyl) as the major cellular fatty acids, ubiquinone Q-8 as a predominant quinone, and diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine as the major polar lipids. A multifaceted investigation demonstrated that strains H21R20T and H23M41T represent novel species of the genus Lysobacter, for which we propose the names Lysobacter ciconiae sp. nov. and Lysobacter avium sp. nov. for strains H21R20T (= KCTC 82316T = JCM 34832T) and H23M41T (= KCTC 62676T = JCM 33223T), respectively. [ABSTRACT FROM AUTHOR]

Cyclic AMP receptor protein (CRP) is a well-characterized group of global transcription factors in bacteria. They are known to regulate numerous cellular processes by binding DNA and/or cAMP (a ligand called bacterial second messenger) to control target gene expression. Gram-negative Lysobacter enzymogenes is a soilborne, plant-beneficial bacterium without flagella that can fight against filamentous fungi and oomycete. Driven by the type IV pilus (T4P) system, this bacterium moves to nearby pathogens and uses a “mobile-attack” antifungal strategy to kill them via heat-stable antifungal factor (HSAF) and abundant lyases. This strategy is controlled by a unique “busy” transcription factor Clp, which is a CRP-like protein that is inactivated by binding of c-di-GMP, another ubiquitous second messenger of bacteria. In this review, we summarize the current progress in how Clp initiates a “mobile-attack” strategy through a series of previously uncharacterized mechanisms, including binding to DNA in a unique pattern, directly interacting with or responding to various small molecules, and interacting specifically with proteins adopting distinct structure. Together, these characteristics highlight the multifunctional roles of Clp in L. enzymogenes, a powerful bacterial warrior against fungal pathogens.

Acyl-homoserine lactone (AHL) is the most studied autoinducer in gram-negative bacteria controlling infections of various pathogens. Quenching of AHL signaling by inhibiting AHL synthesis or AHL-receptor binding via small molecular chemicals or enzymatically degrading AHL is commonly used to block bacterial infections. Here, we describe a new quorum-quenching strategy that directly “acquires” bacterial genes/proteins through a defined platform. We artificially expressed a typical AHL synthase gene pcoI from the biocontrol Pseudomonas fluorescens 2P24 in the antifungal bacterium Lysobacter enzymogenes OH11 lacking AHL production. This step led to the discovery of multiple PcoI interacting protein candidates from L. enzymogenes. The individual expression of these candidate genes in 2P24 led to the identification of Le0959, which encodes leucyl aminopeptidase, an effective protein that inhibits AHL synthesis in 2P24. Therefore, we define Le0959 as LqqP (Lysobacter quorum-quenching protein). The expression of pcoI in E. coli could produce AHL, and the introduction of lqqP into E. coli expressing pcoI could prevent the production of AHL. LqqP directly binds to PcoI, and this protein–protein binding reduced the abundance of free PcoI (capable of AHL synthesis) in vivo, thereby blocking PcoI-dependent AHL production. Overall, this study highlights the discovery of LqqP in quenching AHL quorum sensing by binding to AHL synthase via developing a previously-uncharacterized screening technique for bacterial quorum quenching.

Wool keratin is difficult to degrade as comparing to feathers because of its tough secondary structure. In order to develop an approach for high-value utilization of wool fiber waste by keratinolytic microorganisms, which is produced from shearing, weaving, and industrial processing of wool, screening of wool-degrading bacterium with high degradation efficiency were performed in this study. To this end, Lysobacter brunescens YQ20 was identified and characterized. The optimized conditions for wool degradation were pH 9.0 and 37°C with 20% liquid volume of Erlenmeyer flask. After fermentation, 15 essential amino acids were detected when wool fiber waste was fermented. The total amino acids produced from 1% wool per hour were 13.7 mg/L. The concentration was 8.6-fold higher than that produced by the strain Stenotrophomonas maltophilia BBE11-1, which had previously been reported to have the highest wool-degrading capacity. Our study reports the first Lysobacter strain that exhibits efficient wool degradation and yields higher concentrations of amino acids than previously reported strains. Whole-genome sequencing indicated that there were 18 keratinase-like genes in the genome of YQ20, which exhibited a long evolutionary distance from those of Bacillus. Therefore, L. brunescens YQ20 may have applications in the environmentally friendly management of wool waste as fertilizer in agriculture. [ABSTRACT FROM AUTHOR]

Abstract Small molecules are able to regulate numerous cellular processes through binding to various bacterial receptor proteins, but the mechanisms and functions by which these chemicals coordinate and execute remain poorly understood. 4-hydroxybenzoic acid (4-HBA) and cyclic di-GMP (c-di-GMP) are two such molecules with distinct structures that are produced in Lysobacter enzymogenes to synergistically affect the secretion of an antifungal antibiotic, known as heat-stable antifungal factor (HSAF). In our earlier studies, we showed that CdgL, a YajQ-like protein without DNA-binding domain, was able to physically interact with LysR, a transcription factor, to enhance its binding affinity toward the upstream region of the HSAF biosynthesis operon promoter, hence increasing the HSAF biosynthesis. Interestingly, 4-HBA or c-di-GMP can bind to its cognate receptor of LysR or CdgL, respectively, to regulate the HSAF biosynthesis. Further, c-di-GMP acts by binding to CdgL to induce the dissociation of the CdgL-LysR complex, leading to decreased downstream expression. We now showed that CdgL controlled the transcription of lenB2, which encodes an oxygenase to convert chorismate to 4-HBA. Notably, overexpression of cdgL was found to stimulate lenB2 transcription, which likely increased the intracellular 4-HBA content. Also, 4-HBA could bind to LysR to interrupt the LysR-CdgL complex formation and release of CdgL, which caused a lower affinity of LysR toward DNA and hence decreased HSAF operon expression. These findings, along with our earlier report, allow us to propose a coordination mechanism demonstrating how the HSAF biosynthesis is co-regulated by 4-HBA and c-di-GMP through interactions with their cognate receptors. This new mechanism shall shed light on improving the HSAF yield for practical usage.

Abstract Lysobacter enzymogenes OH11 is a non-flagellated, ubiquitous soil bacterium with broad-spectrum antifungal activities. Although lacking flagella, it employs another type of motile behavior, known as twitching motility that is powered by type IV pilus (T4P) to move towards neighboring crop fungal pathogens to kill them as food. At present, little is known about how this non-flagellated bacterium controls twitching motility that is crucial for its predatory lifestyle. Herein, we present a report on how a non-canonical PilZ domain, PilZLe3639, controls such motility in the non-flagellated L. enzymogenes; it failed to bind with c-di-GMP but seemed to be required for twitching motility. Using bacterial two-hybrid and pull-down approaches, we identified PilBLe0708, one of the PilZLe3639-binding proteins that are essential for the bacterial twitching motility, could serve as an ATPase to supply energy for T4P extension. Through site-mutagenesis approaches, we identified one essential residue of PilZLe3639 that is required for its binding affinity with PilBLe0708 and its regulatory function. Besides, two critical residues within the ATPase catalytic domains of PilBLe0708 were detected to be essential for regulating twitching behavior but not involved in binding with PilZLe3639. Overall, we illustrated that the PilZ-PilB complex formation is indispensable for twitching motility in a non-flagellated bacterium.

Wool keratin is difficult to degrade as comparing to feathers because of its tough secondary structure. In order to develop an approach for high-value utilization of wool fiber waste by keratinolytic microorganisms, which is produced from shearing, weaving, and industrial processing of wool, screening of wool-degrading bacterium with high degradation efficiency were performed in this study. To this end, Lysobacter brunescens YQ20 was identified and characterized. The optimized conditions for wool degradation were pH 9.0 and 37°C with 20% liquid volume of Erlenmeyer flask. After fermentation, 15 essential amino acids were detected when wool fiber waste was fermented. The total amino acids produced from 1% wool per hour were 13.7 mg/L. The concentration was 8.6-fold higher than that produced by the strain Stenotrophomonas maltophilia BBE11-1, which had previously been reported to have the highest wool-degrading capacity. Our study reports the first Lysobacter strain that exhibits efficient wool degradation and yields higher concentrations of amino acids than previously reported strains. Whole-genome sequencing indicated that there were 18 keratinase-like genes in the genome of YQ20, which exhibited a long evolutionary distance from those of Bacillus. Therefore, L. brunescens YQ20 may have applications in the environmentally friendly management of wool waste as fertilizer in agriculture.

Although synthetic pesticides play a major role in plant protection, their application needs to be reduced because of their negative impact on the environment. This applies also to copper preparations, which are used in organic farming. For this reason, alternatives with less impact on the environment are urgently needed. In this context, we evaluated eight isolates of the genus Lysobacter (mainly Lysobacter enzymogenes) for their activity against plant pathogens. In vitro, the investigated Lysobacter isolates showed broad antagonistic activity against several phytopathogenic fungi, oomycetes and bacteria. Enzyme assays revealed diverse activities for the tested isolates. The most promising L. enzymogenes isolate (LEC) was used for further detailed analyses of its efficacy and effective working concentrations. The experiments included in vitro spore and sporangia germination tests and leaf disc assays as well as ad planta growth chamber trials against Alternaria solani and Phytophthora infestans on tomato plants, Pseudoperonospora cubensis on cucumbers and Venturia inaequalis on young potted apple trees. When applied on leaves, dilutions of a culture suspension of LEC had a concentration-dependent, protective effect against the tested pathogens. In all pathosystems tested, the effective concentrations were in the range of 2.5–5% and similarly efficacious to common plant protection agents containing copper hydroxide, wettable sulphur or fenhexamid. Thus, the isolate of L. enzymogenes identified in this study exhibits a broad activity against common plant pathogens and is therefore a promising candidate for the development of a microbial biocontrol agent.

Background: Compounds produced by living organisms serve as an important source of inspiration for the development of pharmaceuticals. A potential source of new natural products are bacteria from a genus with species that are known to produce bioactive natural products, but are relatively understudied. Lysobacter is a genus of bacteria that have attracted attention as possible biocontrol agents and are known to produce antibiotic natural products. To further explore the biosynthetic potential of Lysobacter , we sequenced the genomes of two species and performed genome mining studies on those and publicly available genomes., Results: In this study we produced draft genome sequences for Lysobacter firmicutimachus and Lysobacter yananisis . We additionally examined 113 publicly available Lysobacter genomes and found that biosynthetic potential of individual species ranges broadly, with species having between 1 and nearly 20 biosynthetic gene clusters. Filtering for more complete genome assemblies and 9 or more biosynthetic gene clusters, we performed genome mining on 24 Lysobacter genomes. Within these genomes we identified 21 unique nonribosomal peptide, 11 unique hybrid polyketide/nonribosomal peptide, 4 unique polyketide, and 27 unique lanthipeptide biosynthetic gene clusters that produce uncharacterized compounds. Additionally, we tentatively identified the biosynthetic gene cluster in L. rmicutimachus responsible for producing plusbacins, which has not been previously identified., Conclusions: This study demonstrated that Lysobacter have a large repertoire of natural products that remain to be characterized. Additionally, we found that some Lysobacter species are substantially more biosynthetically gifted than others and that strains of the same species of Lysobacter have similar biosynthetic capacities., Competing Interests: Competing interests The authors declare that they have no competing interests.

This paper reviews the possible biological control of grapevine downy mildew Plasmopara viticola. Often, when the climatic conditions are very favorable for the emergence and manifestation of the disease, the chemical treatment has a low efficiency, this phytopathogen producing significant damage, including as a result of the formation of resistant strains. A modern method of combating it is biological control using microorganisms bacteria and fungi which can inhibit through their presence directly or indirectly the growth and multiplication of the fungus, which we present in this paper. [ABSTRACT FROM AUTHOR]

BackgroundLysobacter species are Gram-negative bacteria widely distributed in soil, plant and freshwater habitats. Lysobacter owes its name to the lytic effects on other microorganisms. To better understand their ecology and interactions with other (micro)organisms, five Lysobacter strains representing the four species L. enzymogenes, L. capsici, L. gummosus and L. antibioticus were subjected to genomics and metabolomics analyses.ResultsComparative genomics revealed a diverse genome content among the Lysobacter species with a core genome of 2,891 and a pangenome of 10,028 coding sequences. Genes encoding type I, II, III, IV, V secretion systems and type IV pili were highly conserved in all five genomes, whereas type VI secretion systems were only found in L. enzymogenes and L. gummosus. Genes encoding components of the flagellar apparatus were absent in the two sequenced L. antibioticus strains. The genomes contained a large number of genes encoding extracellular enzymes including chitinases, glucanases and peptidases. Various nonribosomal peptide synthase (NRPS) and polyketide synthase (PKS) gene clusters encoding putative bioactive metabolites were identified but only few of these clusters were shared between the different species. Metabolic profiling by imaging mass spectrometry complemented, in part, the in silico genome analyses and allowed visualisation of the spatial distribution patterns of several secondary metabolites produced by or induced in Lysobacter species during interactions with the soil-borne fungus Rhizoctonia solani.ConclusionsOur work shows that mining the genomes of Lysobacter species in combination with metabolic profiling provides novel insights into the genomic and metabolic potential of this widely distributed but understudied and versatile bacterial genus.

The rhizosphere is a dynamic region governed by complex microbial interactions where diffusible communication signals produced by bacteria continuously shape the gene expression patterns of individual species and regulate fundamental traits for adaptation to the rhizosphere environment. Lysobacter spp. are common bacterial inhabitants of the rhizosphere and have been frequently associated with soil disease suppressiveness. However, little is known about their ecology and how diffusible communication signals might affect their behavior in the rhizosphere. To shed light on the aspects determining rhizosphere competence and functioning of Lysobacter spp., we carried out a functional and transcriptome analysis on the plant beneficial bacterium Lysobacter capsici AZ78 (AZ78) grown in the presence of the most common diffusible communication signals released by rhizosphere bacteria. Mining the genome of AZ78 and other Lysobacter spp. showed that Lysobacter spp. share genes involved in the production and perception of diffusible signal factors, indole, diffusible factors, and N-acyl-homoserine lactones. Most of the tested diffusible communication signals (i.e., indole and glyoxylic acid) influenced the ability of AZ78 to inhibit the growth of the phytopathogenic oomycete Pythium ultimum and the Gram-positive bacterium Rhodococcus fascians. Moreover, RNA-Seq analysis revealed that nearly 21% of all genes in AZ78 genome were modulated by diffusible communication signals. 13-Methyltetradecanoic acid, glyoxylic acid, and 2,3-butanedione positively influenced the expression of genes related to type IV pilus, which might enable AZ78 to rapidly colonize the rhizosphere. Moreover, glyoxylic acid and 2,3-butanedione downregulated tRNA genes, possibly as a result of the elicitation of biological stress responses. On its behalf, indole downregulated genes related to type IV pilus and the heat-stable antifungal factor, which might result in impairment of twitching motility and antibiotic production in AZ78. These results show that diffusible communication signals may affect the ecology of Lysobacter spp. in the rhizosphere and suggest that diffusible communication signals might be used to foster rhizosphere colonization and functioning of plant beneficial bacteria belonging to the genus Lysobacter.

The rhizosphere is a dynamic region governed by complex microbial interactions where diffusible communication signals produced by bacteria continuously shape the gene expression patterns of individual species and regulate fundamental traits for adaptation to the rhizosphere environment. Lysobacter spp. are common bacterial inhabitants of the rhizosphere and have been frequently associated with soil disease suppressiveness. However, little is known about their ecology and how diffusible communication signals might affect their behavior in the rhizosphere. To shed light on the aspects determining rhizosphere competence and functioning of Lysobacter spp., we carried out a functional and transcriptome analysis on the plant beneficial bacterium Lysobacter capsici AZ78 (AZ78) grown in the presence of the most common diffusible communication signals released by rhizosphere bacteria. Mining the genome of AZ78 and other Lysobacter spp. showed that Lysobacter spp. share genes involved in the production and perception of diffusible signal factors, indole, diffusible factors, and N -acyl-homoserine lactones. Most of the tested diffusible communication signals (i.e., indole and glyoxylic acid) influenced the ability of AZ78 to inhibit the growth of the phytopathogenic oomycete Pythium ultimum and the Gram-positive bacterium Rhodococcus fascians. Moreover, RNA-Seq analysis revealed that nearly 21% of all genes in AZ78 genome were modulated by diffusible communication signals. 13-Methyltetradecanoic acid, glyoxylic acid, and 2,3-butanedione positively influenced the expression of genes related to type IV pilus, which might enable AZ78 to rapidly colonize the rhizosphere. Moreover, glyoxylic acid and 2,3-butanedione downregulated tRNA genes, possibly as a result of the elicitation of biological stress responses. On its behalf, indole downregulated genes related to type IV pilus and the heat-stable antifungal factor, which might result in impairment of twitching motility and antibiotic production in AZ78. These results show that diffusible communication signals may affect the ecology of Lysobacter spp. in the rhizosphere and suggest that diffusible communication signals might be used to foster rhizosphere colonization and functioning of plant beneficial bacteria belonging to the genus Lysobacter. [ABSTRACT FROM AUTHOR]

The lack of functional flagella and the ability to prey upon other microorganisms are well-known traits of Lysobacter enzymogenes, a plant beneficial bacterial species. Here, we report a possible link between these two traits in the model strain L. enzymogenes OH11 (OH11). The genome of OH11 encompasses several homologous genes involved in the flagellum formation but it lacks a functional fliC, encoding the flagellin. Despite the lack of the main component of the flagellum, OH11 genome includes genes involved in the flagellar type III secretion system (FT3SS), which is commonly deployed by flagellated bacteria to transport flagellar subunit proteins. To understand the role played by FT3SS in OH11, we showed that the remaining FT3SS genes were expressed under laboratory conditions. Subsequently, we showed that the identified FT3SS genes involved in the secretion of the hook-capping protein FlgD, suggesting OH11 likely possessed a functional FT3SS system. Blocking FT3SS in OH11 via inactivation of the ATPase FliI impaired the secretion of the proteins Le3970 (protease), Le4493 (ß-1,3-glucanase A) and Le1659 (halo acid dehalogenase family), that showed a toxic activity against the yeast Saccharomyces cerevisiae. The possible link between FT3SS and OH11 antagonism towards S. cerevisiae was also confirmed by loss of toxicity in both mutants of ΔfliI and ΔflhB that lacks the FT3SS structural gene flhB when co-cultured with the yeast strain. The design of synthetic proteins toxic against the Gram-negative bacterium Ralstonia solanacearum further supported the involvement of FT3SS in the ability of OH11 to parasitize other microorganisms. Overall, these results revealed a possible cooption of components of FT3SS system in the competition with other microorganisms in the plant beneficial bacterium OH11 and highlighted a functional divergence of FT3SS between flagellated and non-flagellated bacteria.

Colletotrichum capsici and Fusarium oxysporum are pathogenic fungi that cause anthracnose disease on chili plants (Capsicum annum L.) which can decrease the productivity of chili. The control of the fungus attack is carried out by biocontrol agent of the fungi utilizing chitinase-producing bacteria. Chitinase is an enzyme capable of destroying the fungal cell wall composed of chitin compounds. The aims of the research were to select and to characterize chitinase producing bacteria as biological control agent of Colletotrichum capsici and Fusarium oxysporum. Several methods were carried out in this research, among others: isolation of chitinase bacteria, characterization of chitinase bacteria, measurement of enzyme activity, bacterial antagonistic with pathogenic fungi, observation of fungal hypha damage, and identification of 16S rRNA. The BAE36 and BAD211 isolates are chitinase bacteria isolated from chili plant rhizosphere. The isolate belongs to the Gram-negative bacteria. The inhibitory properties of BAE36 and BAD211 against F. oxysporum and C. capsici are on a scale of 3 (5175%). The percentage of inhibitory of BAE36 isolate was 66.1% capable of inhibiting the growth of C. capsici and 55% capable of inhibiting the growth of F. oxysporum. The hyphae of C. capsici fungus on the antagonistic test showed a thick and the surface of the hyphae was wavy while the F. oxysporum hyphae was thin and looks empty. Identification of isolate with 16S rRNA described that 99% of BAE36 and BAD211 isolates were similar to Lysobacter enzymogenes. Precipitation of chitinase enzyme of Lysobacter enzymogenes with 30% ammonium sulphate could increase the enzyme activity. The enzyme precipitation could inhibit the growth of mycelia fungi C. capsici and F. oxysporum.

In the soil gammaproteobacterium Lysobacter enzymogenes, a natural fungal predator, the response regulator PilR controls type IV pili (T4P)‐mediated twitching motility as well as synthesis of the heat‐stable antifungal factor (HSAF). Earlier we showed that PilR acts via the second messenger, c‐di‐GMP; however, the mechanism remained unknown. Here, we describe how PilR, c‐di‐GMP signalling, and HSAF synthesis are connected. We screened genes for putative diguanylate cyclases (c‐di‐GMP synthases) and found that PilR binds to the promoter region of lchD and down‐regulates its transcription. The DNA‐binding affinity of PilR, and therefore its repressor function, are enhanced by phosphorylation by its cognate histidine kinase, PilS. The lchD gene product is a diguanylate cyclase, and the decrease in LchD levels shifts the ratio of c‐di‐GMP‐bound and c‐di‐GMP‐free transcription factor Clp, a key activator of the HSAF biosynthesis operon expression. Furthermore, Clp directly interacts with LchD and enhances its diguanylate cyclase activity. Therefore, the PilS–PilR two‐component system activates T4P‐motility while simultaneously decreasing c‐di‐GMP levels and promoting HSAF production via the highly specific LchD–c‐di‐GMP–Clp pathway. Coordinated increase in motility and secretion of the "long‐distance" antifungal weapon HSAF is expected to ensure safer grazing of L. enzymogenes on soil or plant surfaces, unimpeded by fungal competitors, or to facilitate bacterial preying on killed fungal cells. This study uncovered the mechanism of coregulated pili‐based motility and production of an antifungal antibiotic in L. enzymogenes, showcased the expanded range of functions of the PilS–PilR system, and highlighted exquisite specificity in c‐di‐GMP‐mediated circuits. [ABSTRACT FROM AUTHOR]

In the present study, exopolysaccharide (EPS) produced by Lysobacter sp. MMG2 (lyEPS) was characterized and purified. The lyEPS-producing strain Lysobacter sp. MMG2 was isolated from the roots of Tagetes patula. When lyEPS was produced in tryptic soy broth with 1% glucose and the lyophilized powder was measured, the yield was found to be 0.67 g/L. The molecular weight (Mw) of lyEPS was 1.01 × 105 Da. Its monosaccharide composition includes 84.24% mannose, 9.73% glucose, 2.55% galactose, 2.77% arabinose, 0.32% xylose, and 0.03% rhamnose. Scanning electron microscopy (SEM) revealed that lyEPS has various round and rough surfaces. Fourier-transform infrared (FTIR) analysis identified its carbohydrate polymer functional groups. Moreover, thermogravimetric analysis of lyEPS revealed two events of mass loss: the first was water loss, which resulted in 3.97% mass loss and the second event occurred at approximately 212 °C. lyEPS could inhibit biofilm-producing pathogenic bacteria without any antimicrobial activity. Furthermore, lyEPS at a concentration of 4 mg/mL could exhibit potent 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical-scavenging activity (89.25%). These results indicate that lyEPS could be a promising candidate for industrial development if its biological activity is further explored.

The chitin-assimilating gram-negative bacterium, Lysobacter sp. MK9-1, was isolated from soil and was the source of a glycoside hydrolase family 19-type chitinase (Chi19MK) gene that is 933-bp long and encodes a 311-residue protein. The deduced amino acid sequence of Chi19MK includes a signal peptide, an uncharacterized sequence, a carbohydrate-binding module family 12-type chitin binding domain, and a catalytic domain. The catalytic domain of Chi19MK is approximately 60% similar to those of ChiB from Burkholderia gladioli CHB101, chitinase N (ChiN) from Chitiniphilus shinanonensis SAY3T, ChiF from Streptomyces coelicolor A3(2), Chi30 from Streptomyces olivaceoviridisis , ChiA from Streptomyces cyaneus SP-27, and ChiC from Streptomyces griseus HUT6037. Chi19MK lacking the signal and uncharacterized sequences (Chi19MKΔNTerm) was expressed in Escherichia coli Rosetta-gami B(DE3), resulting in significant chitinase activity in the soluble fraction. Purified Chi19MKΔNTerm hydrolyzed colloidal chitin and released disaccharide. Furthermore, Chi19MKΔNTerm inhibited hyphal extension in Trichoderma reesei and Schizophyllum commune. Based on quantitative antifungal activity assays, Chi19MKΔNTerm inhibits the growth of Trichoderma viride with an IC 50 value of 0.81 μM. [ABSTRACT FROM AUTHOR]

Siderophores are produced by bacteria in iron-restricted conditions. However, we found maltose could induce the biosynthesis of the siderophore lysochelin in Lysobacter sp. 3655 in rich media that are not compatible with siderophore production. Maltose markedly promoted cell growth, with over 300% increase in cell density (OD 600 ) when LB medium was added with maltose (LBM). While lysochelin was not detectable when OD 600 in LBM was below 5.0, the siderophore was clearly produced when OD 600 reached 7.5 and dramatically increased when OD 600 was 15.0. Coincidently, the transcription of lysochelin biosynthesis genes was remarkably enhanced following the increase of OD 600 . Conversely, the iron concentration in the cell culture dropped to 1.2 μM when OD 600 reached 15.0, which was 6-fold lower than that in the starting medium. Moreover, mutants of the maltose-utilizing genes ( orf2677 and orf2678 ) or quorum-sensing related gene orf644 significantly lowered the lysochelin yield. Transcriptomics analysis showed that the iron-utilizing/up-taking genes were up-regulated under high cell density. Accordingly, the transcription of lysochelin biosynthetic genes and the yield of lysochelin were stimulated when the iron-utilizing/up-taking genes were deleted. Finally, lysochelin biosynthesis was positively regulated by a TetR regulator (ORF3043). The lysochelin yield in orf3043 mutant decreased to 50% of that in the wild type and then restored in the complementary strain. Together, this study revealed a previously unrecognized mechanism for lysochelin biosynthetic regulation, by which the siderophore could still be massively produced in Lysobacter even grown in a rich culture medium. This finding could find new applications in large-scale production of siderophores in bacteria., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zhang, Liu, Jiang, Zheng, Yu and Du.)

Two Gram-negative bacteria, designated as strains LF1 T and HM2-2 T , were isolated from an artificial pond in a honey farm at Hoengseong-gun, Gangwon-do, Republic of Korea. The 16S rRNA sequence analysis results revealed that strain LF1 T belonged to the genus Lysobacter and had the highest sequence similarity to Lysobacter niastensis GH41-7 T (99.0 %), Lysobacter panacisoli CJ29 T (98.9 %), and Lysobacter prati SYSU H10001 T (98.2 %). Its growth occurred at 20-37 °C, at pH 5.0-12.0, and in the presence of 0-2% NaCl. The major fatty acids were iso-C 15 : 0 , iso-C 16 : 0 , and summed feature 9 (iso-C 17 : 1  ω 9 c and/or C 16 : 0 10-methyl). The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. The DNA G+C content was 67.5 mol%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain LF1 T and species of the genus Lysobacter were 79.1-84.4% and 22.0-27.5 %, respectively. The 16S rRNA sequence analysis results revealed that strain HM2-2 T belonged to the genus Limnohabitans and was most closely related to Limnohabitans planktonicus II-D5 T (98.9 %), Limnohabitans radicicola JUR4 T (98.4%), and Limnohabitans parvus II-B4 T (98.4 %). Its growth occurred at 10-35 °C, at pH 5.0-11.0, and in the presence of 0-2% NaCl. The major fatty acids were C 16 : 0 and summed feature 3 (C 16 : 1  ω 7 c /C 16 : 1  ω 6 c ). The major polar lipid was phosphatidylethanolamine. The DNA G+C content was 59.9 mol%. The ANI and dDDH values between strain HM2-2 T and its closely related strains were 75.1-83.0% and 20.4-26.4 %, respectively. Phenotypic, genomic, and phylogenetic data revealed that strains LF1 T and HM2-2 T represent novel species in the genera Lysobacter and Limnohabitans , for which the names Lysobacter stagni sp. nov. and Limnohabitans lacus sp. nov. are proposed, respectively. The type strain of Lys. stagni is LF1 T (=KACC 23251 T =TBRC 17648 T ), and that of Lim. lacus is HM2-2 T (=KACC 23250 T =TBRC 17649 T ).

Soil microorganisms play a crucial role in nutrient availability and overall soil health. However, the effects that biochar has on soil microbial communities are not well understood. This study analysed the effects of biochar pyrolysis temperature and application rate on the soil microbial community of loess. Two biochars derived from soybean stover were produced at 300 and 600°C (BC300 and BC600, respectively) and were applied to loess at the rates of 1, 3, and 5% (w/w). After fifteen weeks of incubation, soil microbial analysis was performed using 16S rDNA amplicon sequencing technology. All of the BC300 and BC600 treated soils were shown to have an increase in the relative abundance of Gemmatimonadetes and a decrease in Actinobacteria and Chloroflexi. Proteobacteria also showed a significant increase in the majority of the biochar treated soils. Biochar led to a shift in the soil microbial community and caused a significant increase in the relative abundance of bacteria from the genus Lysobacter. Based on the results of this study, soybean stover-derived biochar should be considered as a potential soil amendment for improving the health of loess or other soils in semi-arid climates. [ABSTRACT FROM AUTHOR]

It has become increasingly recognized that hormesis phenomena exist in soil ecosystem, but the research on the hormetic responses of soil enzymes are still limited. This study was conducted to investigate the hormetic effects of lead (Pb) on the activity of soil alkaline phosphatase (ALP) and the associated microbial groups. Soils were treated by adding Pb (NO3)2 solution with 0, 10, 100, 500, 1000, 2000, 4000, and 5000 mg/kg of Pb, respectively. A moist heat sterilization method (121 °C × 30 min) was used to discriminate the microbial effect on soil ALP hormesis from other factors. The bacterial community composition and abundance in the control (CK) and Pb-treated soils were detected by the high-throughput sequencing technique. The ALP activity at doses of 500–1000 mg/kg of Pb was significantly higher than that of CK (0 mg/kg of Pb), showing a typical inverted U-shaped dose response with the stimulation magnitude of 9.8–10.3% within 48 h of incubation. In addition, ALP activity decreased by 80% on average after soil sterilization. Analysis of bacterial community composition indicated that the relative abundance of Lysobacter at 1000 mg Pb/kg was higher than that of CK at genus level, with the increase of 69.82%. The highly significant correlation between soil ALP activities and relative abundance of Lysobacter indicated that this bacterial genus could possibly contribute to the hormetic responses of soil ALP to added doses of Pb in soils. [ABSTRACT FROM AUTHOR]

A bacterial strain isolated from a soil collected in Jeju Island, designated as 17J7-1T, was Gram-negative, rod-shaped, yellow colored, and motile by gliding. This strain was able to grow at temperature range from 10 to 42 °C, pH 7–9, and tolerated up to 1% NaCl. Analysis of 16S rRNA sequence identified strain 17J7-1T as a member of the genus Lysobacter with close sequence similarity with Lysobacter mobilis 9NM-14T (97.4%), Lysobacter xinjiangensis RCML-52T (97.0%), and Lysobacter humi FJY8T (96.9%). The genomic DNA G + C content of the isolate was 67.9 mol%. DNA–DNA relatedness between strain 17J7-1T and L. mobilis, L. humi, and L. xinjiangensis were 42.3%, 39.5%, and 35.8%, respectively, clearly showing that the isolate is distinct from its closest phylogenetic neighbors in the genus Lysobacter. Average nucleotide identity (ANI) and digital DNA–DNAhybridization (dDDH) values between strain 17J7-1T and L. enzymogenes ATCC 29487T, the type species of this genus, and several other close Lysobacter species were less than 77% and 22%, respectively. Major fatty acids were C16:0 iso (29.8%), summed feature 9 (C17:1 iso ω9c/C16:0 10-methyl; 20.1%), and C15:0 iso (17.7%). The predominant respiratory quinone was ubiquinone Q-8 and the major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. In the light of the polyphasic evidence accumulated in this study, strain 17J7-1T is considered to represent a novel species in the genus Lysobacter, for which name Lysobacter terrigena sp. nov. is proposed. The type strain is 17J7-1T (= KCTC 62217T = JCM 33057T). [ABSTRACT FROM AUTHOR]

Summary: YajQ, a binding protein of the universal bacterial second messenger cyclic di‐GMP (c‐di‐GMP), affects virulence in several bacterial pathogens, including Xanthomonas campestris. In this bacterium, YajQ interacts with the transcription factor LysR. Upon c‐di‐GMP binding, the whole c‐di‐GMP‐YajQ‐LysR complex is found to dissociate from DNA, resulting in virulence gene regulation. Here, we identify a YajQ‐LysR‐like system in the bacterial biocontrol agent Lysobacter enzymogenes OH11 that secretes an antifungal antibiotic, heat‐stable antifungal factor (HSAF) against crop fungal pathogens. We show that the YajQ homologue, CdgL (c‐di‐GMP receptor interacting with LysR) affects expression of the HSAF biosynthesis operon by interacting with the transcription activator LysR. The CdgL‐LysR interaction enhances the apparent affinity of LysR to the promoter region upstream of the HSAF biosynthesis operon, which increases operon expression. Unlike the homologues CdgL (YajQ)‐LysR system in X. campestris, we show that c‐di‐GMP binding to CdgL seems to weaken CdgL‐LysR interactions and promote the release of CdgL from the LysR‐DNA complex, which leads to decreased expression. Together, this study takes the YajQ‐LysR‐like system from bacterial pathogens to a crop‐protecting bacterium that is able to regulate antifungal HSAF biosynthesis via disassembly of the c‐di‐GMP receptor–transcription activator complex. [ABSTRACT FROM AUTHOR]

Discovery of multidrug resistance (MDR) in environmental microorganisms provides unique resources for uncovering antibiotic resistomes, which could be vital to predict future emergence of MDR pathogens. Our previous studies indicated that Lysobacter sp. conferred intrinsic resistance to multiple antibiotics at high levels, especially ampicillin, the first broad-spectrum β-lactam antibiotics against both Gram-positive and Gram-negative bacteria. However, the underlying molecular mechanisms for resistance to ampicillin in Lysobacter enzymogenes strain C3 (LeC3) remain unknown. In this study, screening a Tn5 transposon mutant library of LeC3 recovered 12 mutants with decreased ampicillin resistance, and three mutants (i.e., tatC, lebla, and lpp) were selected for further characterization. Our results revealed that genes encoding β-lactamase (lebla) and twin-arginine translocation (tatC) system for β-lactamase transport played a pivotal role in conferring ampicillin resistance in L. enzymogenes. It was also demonstrated that the lpp gene was not only involved in resistance against β-lactams but also conferred resistance to multiple antibiotics in L. enzymogenes. Permeability assay results indicated that decreased MDR in the lpp mutant was in part due to its higher cellular permeability. Furthermore, our results showed that the difference of LeC3 and L. antibioticus strain LaATCC29479 in ampicillin susceptibility was partly due to their differences in cellular permeability, but not due to β-lactamase activities. [ABSTRACT FROM AUTHOR]

Abstract Background The environmental gliding bacteria Lysobacter are emerging as a new group of biocontrol agents due to their prolific production of lytic enzymes and potent antibiotic natural products. These bacteria are intrinsically resistant to many antibiotics, but the mechanisms behind the antibiotic resistance have not been investigated. Results Previously, we have used chloramphenicol acetyltransferase gene (cat) as a selection marker in genetic manipulation of natural product biosynthetic genes in Lysobacter, because chloramphenicol is one of the two common antibiotics that Lysobacter are susceptible to. Here, we found L. enzymogenes, the most studied species of this genus, could still grow in the presence of a low concentration of chloramphenicol. Three chloramphenicol derivatives (1–3) with an unusual acylation pattern were identified in a cat-containing mutant of L. enzymogenes and in the wild type. The compounds included chloramphenicol 3'-isobutyrate (1), a new compound chloramphenicol 1'-isobutyrate (2), and a rare chloramphenicol 3'-isovalerate (3). Furthermore, a mutation of a global regulator gene (clp) or a Gcn5-related N-acetyltransferase (GNAT) gene in L. enzymogenes led to nearly no growth in media containing chloramphenicol, whereas a complementation of clp restored the chloramphenicol acylation as well as antibiotic HSAF production in the clp mutant. Conclusions The results indicated that L. enzymogenes contains a pool of unusual acyl donors for enzymatic modification of chloramphenicol that confers the resistance, which may involve the Clp-GNAT regulatory system. Because Lysobacter are ubiquitous inhabitants of soil and water, the finding may have important implications in understanding microbial competitions and bioactive natural product regulation.

Abstract Ax21 family proteins have been shown to play regulatory roles in plant- and animal-pathogenic species in the bacterial family Xanthomonadaceae, but the protein have not been investigated previously in the non-pathogenic members of this bacterial family. Lysobacter enzymogenes, is a non-pathogenic species known for its capacity as a biocontrol agent of plant pathogens. It is also noted for the production of antimicrobial secondary metabolites, heat stable antifungal factor (HSAF) and WAP-8294A2, that have potential for agricultural and pharmaceutical applications. The species also displays type IV pili-dependent twitching motility and the production of multiple extracellular lytic enzymes as additional biocontrol-related traits. Here, we show that L. enzymogenes strain OH11 possesses three genes widely separated in the OH11 genome that code for unique Ax21-like proteins (Lsp). By comparing the wildtype OH11 with mutant strains having a single lsp gene or a combination of lsp genes deleted, we found that each Lsp protein individually is involved in positive regulation of HSAF and WAP-8294A2 biosynthesis, but the proteins collectively do not exert additive effects in this regulation. None of the Lsp proteins were found to influence twitching motility or the production of three extracellular lytic enzymes. This study is the first to provide evidence linking Ax21-family proteins to antibiotic biosynthesis and, hence, adds new insights into the diversity of regulatory functions of Ax21 family proteins in bacteria.

Deoxynivalenol (DON) is a widely distributed mycotoxin that frequently occurs in various agricultural raw materials and feeds. DON acts as a virulence factor that accelerates the spread of plant diseases; moreover, its accumulation in grains causes yield loss and serious health problems to humans and livestock. Biodegradation of DON into less- or non-toxic substances using naturally existing microorganisms is considered the best approach for DON detoxification. Although various single isolates and mixed cultures capable of detoxifying DON have been reported, details of the metabolic pathways and the degrading enzymes/coding genes involved are scarce. In this study, we aimed to isolate DON-degrading bacteria from soil samples and explore the mechanisms. Toward this end, 85 soil samples collected from different provinces in China were enriched under aerobic conditions with mineral media containing 50 μg/ml of DON as the sole carbon source. The bacterial consortium LZ-N1 exhibited highly efficient and steady DON-transforming activity. High-throughput sequencing was used to characterize the composition of the involved microflora, and analysis of 16S rRNA sequences indicated that LZ-N1 was composed of at least 11 bacterial genera, with Pseudomonas accounting for nearly half the relative abundance. Coincubation of a mixed culture of two novel strains from the LZ-N1 consortium, namely Pseudomonas sp. Y1 and Lysobacter sp. S1, showed sustained transformation of DON into the metabolite 3-epi-deoxynivalenol, with no degradation products detected after 72 h. The cell-free supernatant, lysate, and cell debris of the mixed culture possessed DON-degrading ability, with the supernatant reaching a DON degradation rate of 100% within 48 h with 50 μg/ml of DON. This is the first report of two-step enzymatic epimerization of DON by a mixed culture, which may provide a new insight into this pathway for future applications in detoxification of DON-contaminated cereals and feed.