Lysobacter arvi sp. nov. Isolated from Farmland Soil.

A bacterial strain designated as UC was isolated from farmland soil. Strain UCT formed a pale yellow colony on nutrient agar. Cell morphology revealed it as the rod-shaped bacterium that stained Gram-negative. The 16S rRNA gene sequence analysis identified strain UCT as a member of the genus Lysobacter that showed high identity with L. soli DCY21T (99.5%), L. panacisoli CJ29T (98.7%), and L. tabacisoli C8-1T (97.9%). It formed a distinct cluster with these strains in the neighbor-joining phylogenetic tree. A similar tree topology was observed in TYGS-based phylogenomic analysis. However, genome sequence analyses of strain UCT showed 87.7% average nucleotide identity and 34.7% digital DNA-DNA hybridization similarity with the phylogenetically closest species, L. soli DCY21T. The similarity was much less with other closely related strains of the genus Lysobacter. The G + C content of strain UCT was 68.1%. Major cellular fatty acids observed were C14:0 iso (13.4%), C15:0 iso (13.6%), and C15:0 anteiso (14.8%). Quinone Q-8 was the major respiratory ubiquinone. Predominant polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, and phosphatidylglycerol. Production of xanthomonadin pigment was observed. Based on phenotypic differences and phylogenomic analysis, strain UCT represents a novel species of the genus Lysobacter, for which the name Lysobacter arvi is proposed. The type strain of the novel species is UCT (= KCTC 92613T = JCM 23757T = MTCC 12824T).

The Transcription Regulator GntR/HutC Regulates Biofilm Formation, Motility and Stress Tolerance in Lysobacter capsici X2-3.

Lysobacter capsici X2-3, a plant growth-promoting rhizobacteria (PGPR), was isolated from wheat rhizosphere and has inhibitory effects against a wide range of pathogens. One important characteristic of L. capsici is its ability to produce diverse antibiotics and lytic enzymes. The GntR family of transcription factors is a common transcription factor superfamily in bacteria that has fundamental roles in bacterial metabolism regulation. However, the GntR family transcription factor in Lysobacter has not been identified. In this study, to obtain an understanding of the GntR/HutC gene function in L. capsici X2-3, a random Tn5-insertion mutant library of X2-3 was constructed to select genes showing pleiotropic effects on phenotype. We identified a Tn5 mutant with an insertion in LC4356 that showed reduced biofilm levels, and sequence analysis indicated that the inserted gene encodes a GntR/HutC family transcription regulator. Furthermore, the LC4356 mutant showed reduced extracellular polysaccharide (EPS) production, diminished twitching motility and decreased survival under UV radiation and high-temperature. The RT‒qPCR results indicated that the pentose phosphate pathway-related genes G6PDH, 6PGL and PGDH were upregulated in the LC4356 mutant. Thus, since L. capsici is an efficient biocontrol agent for crop protection, our findings provide fundamental insights into GntR/HutC and will be worthwhile to improve PGPR biocontrol efficacy.

Lysohexaenetides A and B, linear lipopeptides from Lysobacter sp. DSM 3655 identified by heterologous expression in Streptomyces.

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.

Lysobacter changpingensis sp. nov., a novel species of the genus Lysobacter isolated from a rhizosphere soil of strawberry in China.

In the present work, we characterized in detail strain CM-3-T8T, which was isolated from the rhizosphere soil of strawberries in Beijing, China, in order to elucidate its taxonomic position. Cells of strain CM-3-T8T were Gram-negative, non-spore-forming, aerobic, short rod. Growth occurred at 25-37 °C, pH 5.0-10.0, and in the presence of 0-8% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain CM-3-T8T formed a stable clade with Lysobacter soli DCY21T and Lysobacter panacisoli CJ29T, with the 16S rRNA gene sequence similarities of 98.91% and 98.50%. The average nucleotide identity and digital DNA-DNA hybridization values between strain SG-8 T and the two reference type strains listed above were 76.3%, 79.6%, and 34.3%, 27%, respectively. The DNA G + C content was 68.4% (mol/mol). The major cellular fatty acids were comprised of C15:0 iso (36.15%), C17:0 iso (8.40%), and C11:0 iso 3OH (8.28%). The major quinone system was ubiquinone Q-8. The major polar lipids were phosphatidylethanolamine (PE), phosphatidylethanolamine (PME), diphosphatidylglycerol (DPG), and aminophospholipid (APL). On the basis of phenotypic, genotypic, and phylogenetic evidence, strain CM-3-T8T (= ACCC 61714 T = JCM 34576 T) represents a new species within the genus Lysobacter, for which the name Lysobacter changpingensis sp. nov. is proposed.

Identification and Biosynthetic Study of the Siderophore Lysochelin in the Biocontrol Agent Lysobacter enzymogenes.

Lysobacter is a genus of bacteria emerging as new biocontrol agents in agriculture. Although iron acquisition is essential for the bacteria, no siderophore has been identified from any Lysobacter. Here, we report the identification of the first siderophore, N1,N8-bis(2,3-dihydroxybenzoyl)spermidine (lysochelin), and its biosynthetic gene cluster from Lysobacter enzymogenes. Intriguingly, the deletion of the spermidine biosynthetic gene encoding arginine decarboxylase or SAM decarboxylase eliminated lysochelin and the antifungals, HSAF and its analogues, which are key to the disease control activity and to the survival of Lysobacter under oxidative stresses caused by excess iron. The production of lysochelin and the antifungals is greatly affected by iron concentration. Together, the results revealed a previously unrecognized system, in which L. enzymogenes produces a group of small molecules, lysochelin, spermidine, and HSAF and its analogues, that are affected by iron concentration and critical to the growth and survival of the biocontrol agent.

Physiological and genomic analyses of cobalamin (vitamin B12)-auxotrophy of Lysobacter auxotrophicus sp. nov., a methionine-auxotrophic chitinolytic bacterium isolated from chitin-treated soil.

A novel bacterium, designated 5-21aT, isolated from chitin-treated upland soil, exhibits methionine (Met) auxotrophy and chitinolytic activity. A physiological experiment revealed the cobalamin (synonym, vitamin B12)(Cbl)-auxotrophic property of strain 5-21aT. The newly determined complete genomic sequence indicated that strain 5-21aT possesses only the putative gene for Cbl-dependent Met synthase (MetH) and lacks that for the Cbl-independent one (MetE), which implies the requirement of Cbl for Met-synthesis in strain 5-21aT. The set of genes for the upstream (corrin ring synthesis) pathway of Cbl synthesis is absent in the genome of strain 5-21aT, which explains the Cbl-auxotrophy of 5-21aT. This strain was characterized via a polyphasic approach to determine its taxonomic position. The nucleotide sequences of two copies of the 16S rRNA gene of strain 5-21aT indicated the highest similarities to Lysobacter soli DCY21T(99.8 and 99.9 %) and Lysobacter panacisoli CJ29T(98.7 and 98.8 %, respectively), whose Cbl-auxotrophic properties were revealed in this study. The principal respiratory quinone was Q-8. The predominant cellular fatty acids were iso-C15:0, iso-C16:0 and iso-C17:1 ω9c. The complete genome sequence of strain 5-21aT revealed that the genome size was 4 155 451 bp long and the G+C content was 67.87 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between strain 5-21aT and its most closely phylogenetic relative L. soli DCY21T were 88.8 and 36.5%, respectively. Based on genomic, chemotaxonomic, phenotypic and phylogenetic data, strain 5-21aT represents a novel species in the genus Lysobacter, for which the name Lyobacter auxotrophicus sp. nov. is proposed. The type strain is 5-21aT (=NBRC 115507T=LMG 32660T).

The posttranscriptional regulator CsrA affects multidrug resistance and biocontrol activity in Lysobacter enzymogenes.

AIMS: The posttranscriptional regulator CsrA regulates many cellular processes, including stress responses in diverse bacteria. However, the role of CsrA in multidrug resistance (MDR) and biocontrol activity in Lysobacter enzymogenes strain C3 (LeC3) remains unknown. METHODS AND RESULTS: In this study, we demonstrated that deletion of the csrA gene resulted in the initial slow growth of LeC3 and reduced its resistance to multiple antibiotics, including nalidixic acid (NAL), rifampicin (RIF), kanamycin (Km), and nitrofurantoin (NIT). Loss of the csrA gene also reduced its ability in inhibiting hypha growth of Sclerotium sclerotiorum and influenced its extracellular cellulase and protease activities. Two putative small noncoding regulatory RNAs (sRNAs), referred to as csrB and csrC, were also revealed in the genome of LeC3. Double deletion of csrB and csrC in LeC3 led to increased resistance to NAL, RIF, Km, and NIT. However, no difference was observed between LeC3 and the csrB/csrC double mutant in their suppression of S. sclerotiorum hypha growth and production of extracellular enzymes. CONCLUSION: These results suggest that CsrA in LeC3 not only conferred its intrinsic MDR, but also contributed to its biocontrol activity.

Twitching Motility Assays of Lysobacter enzymogenes OH11 Under a Light Microscope.

Bacterial twitching motility is a peculiar way of adherence and surface translocation on moist solid or semisolid surfaces. Although the twitching motility has been detected in various flagellated bacteria, such as Pseudomonas aeruginosa, it has been rarely detected in flagella-less bacteria like Lysobacter enzymogenes, a natural predator of filamentous fungi. Here, by using a strain OH11 of L. enzymogenes as a model system, we describe a convenient method for observing the twitching motility, with fewer steps and better repetition than conventional methods. This new method provides important technical support for the motile study of Lysobacter.

Lysohexaenetides A and B, linear lipopeptides from Lysobacter sp. DSM 3655 identified by heterologous expression in Streptomyces / 中国天然药物

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.

The Wsp chemosensory system modulates c-di-GMP-dependent biofilm formation by integrating DSF quorum sensing through the WspR-RpfG complex in Lysobacter.

The ubiquitous Wsp (wrinkly spreader phenotype) chemosensory system and DSF (diffusible signal factor) quorum sensing are two important chemically associated signaling systems that mediate bacterial communications between the host and environment. Although these two systems individually control biofilm formation in pathogenic bacteria via the ubiquitous second messenger c-di-GMP, their crosstalk mechanisms remain elusive. Here we present a scenario from the plant-beneficial and antifungal bacterium Lysobacter enzymogenes OH11, where biofilm formation favors the colonization of this bacterium in fungal hyphae. We found that the Wsp system regulated biofilm formation via WspR-mediated c-di-GMP signaling, whereas DSF system did not depend on the enzymatic activity of RpfG to regulate biofilm formation. We further found that WspR, a diguanylate cyclase (DGC) responsible for c-di-GMP synthesis, could directly bind to one of the DSF signaling components, RpfG, an active phosphodiesterase (PDE) responsible for c-di-GMP degradation. Thus, the WspR-RpfG complex represents a previously undiscovered molecular linker connecting the Wsp and DSF systems. Mechanistically, RpfG could function as an adaptor protein to bind and inhibit the DGC activity of unphosphorylated WspR independent of its PDE activity. Phosphorylation of WspR impaired its binding affinity to RpfG and also blocked the ability of RpfG to act as an adaptor protein, which enabled the Wsp system to regulate biofilm formation in a c-di-GMP-dependent manner by dynamically integrating the DSF system. Our findings demonstrated a previously uncharacterized mechanism of crosstalk between Wsp and DSF systems in plant-beneficial and antifungal bacteria.

Lysobacter lactosilyticus sp. nov., a Novel ß-Galactosidase Producing Bacterial Strain Isolated from Farmland Soil Applied with Amino Acid Fertilizer.

To isolate ß-galactosidase producing bacterial resources, a novel Gram-stain-negative, strictly aerobic bacterial strain designated as A6T was obtained from a farmland soil sample. Cells of the strain were rod-shaped (0.4-0.7 µm × 1.8-2.2 µm) without flagella and motility. Strain A6T grew optimally at 30 °C, pH 7.0 with 0% (w/v) NaCl. Based on phylogenetic analysis, strain A6T clustered within the genus Lysobacter clade and branched with Lysobacter dokdonensis KCTC 12822T (99.5%, 16S rRNA gene sequence similarity) and Lysobacter caseinilyticus KACC 19816T (98.5%). The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain A6T and Lysobacter dokdonensis KCTC 12822T were 82.7% and 26.2%, and the values for strain A6T and KACC 19816T were 81.4% and 23.8%, respectively. Iso-C16:0, iso-C15:0, summed feature 9 (C17:1 iso ω9c and/or C16:0 10-methyl) and summed feature 3 (C16:1ω7c and/or C16:1 ω6c) were the major fatty acids, diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine were the major polar lipids, and ubiquinone 8 (Q-8) was the major ubiquinone. The genomic DNA G+C content was 67.2 mol%. Furthermore, under the condition of 30 °C, pH 7.0, 4% inoculation with 10.0 g L-1 lactose, the ß-galactosidase activity produced by strain A6T was highest, reaching 95.3 U mL-1, indicating that this strain could be applied as a potential strain for ß-galactosidase production. Strain A6T represents a novel species of the genus Lysobacter, and Lysobacter lactosilyticus sp. nov. is proposed on the basis of phenotypic, genotypic, and chemotaxonomic analysis. The type strain is A6T (=KCTC 82184T=CGMCC 1.18582T).

Lysobacter sedimenti sp. nov., Isolated from the Sediment, and Reclassification of Luteimonas lumbrici as Lysobacter lumbrici comb. nov.

A bacterium, designated 50T was isolated from the sediment of a pesticide plant in Shandong Province, PR China. The strain was non-motile, Gram stain-negative, rod shaped and grew optimally on NA medium at 30 °C, pH 7.5 and with 0% (w/v) NaCl. Strain 50T showed the highest 16S rRNA gene sequence similarity with Lysobacter pocheonensis Gsoil 193T (96.7%), followed by Luteimonas lumbrici 1.1416T (96.5%). Phylogenetic analyses based on 16S rRNA indicated that strain 50T and Luteimonas lumbrici 1.1416T were clustered with the genus of Lysobacter and formed a subclade with Lysobacter pocheonensis Gsoil 193T. In the phylogenetic analysis based on the genome sequences, strain 50T and Luteimonas lumbrici 1.1416T were also clustered with the type strains of the genus Lysobacter. The obtained ANI and the dDDH value between 50T and Luteimonas lumbrici 1.1416T were 80.6% and 24.0%, respectively. The respiratory quinone was ubiquinone-8 (Q-8), and the major cellular fatty acids were iso-C15: 0 (31.7%), summed feature 9 (iso-C17:1 ω9c or C16:0 10-methyl) (23.7%), iso-C17:0 (14.3%) and iso-C16:0 (12.6%). The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and unidentified aminophospholipid, unidentified phospholipid and unidentified lipid. The genomic DNA G + C content was 69.5 mol%. According to the phenotypic, chemotaxonomic and phylogenetic analyses, strain 50T represents a novel species of the genus Lysobacter, for which the name Lysobacter sedimenti sp. nov. is proposed, with strain 50T (= KCTC 92088T = CCTCC AB 2022035T) as the type strain. In this study, it is also proposed that Luteimonas lumbrici should be transferred to the genus Lysobacter as Lysobacter lumbrici comb. nov. The type strain of Lysobacter lumbrici is 1.1416T (= KCTC 62979T = CCTCC AB 2018348T).

Lysobacter selenitireducens sp. nov., isolated from river sediment.

A Gram-stain-negative, yellow-pigmented, motile, flagellated and rod-shaped bacterium, designated as 13AT, was isolated from a river sediment sample of Fuyang River in Hengshui City, Hebei Province, PR China. Strain 13AT grew at 10-37 °C (optimum, 30 °C), at pH 5.0-11.0 (optimum, pH 7.0) and at 0-7 % (w/v) NaCl concentration (optimum, 0 %). Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain 13AT belongs to the genus Lysobacter, and was most closely related to Lysobacter spongiicola DSM 21749T (97.8 %), Lysobacter concretionis DSM 16239T (97.5 %), Lysobacter daejeonensis GIM 1.690T (97.3 %) and Lysobacter arseniciresistens CGMCC 1.10752T (96.9 %). Meanwhile, the type species Lysobacter enzymogenes ATCC 29487T was selected as a reference strain (95.2 %). The genomic size of strain 13AT was 3.0 Mb and the DNA G+C content was 69.0 %. The average nucleotide identity values between strain 13AT and each of the reference type strains L. spongiicola DSM 21749T, L. concretionis DSM 16239T, L. daejeonensis GIM 1.690T, L. arseniciresistens CGMCC 1.10752T and L. enzymogenes ATCC 29487T were 75.9, 76.1, 77.7, 78.0 and 73.2 %, respectively. The digital DNA-DNA hybridization values between strain 13AT and each of the reference type strains were 21.7, 22.2, 21.9, 22.7 and 23.2 %, respectively. The average amino acid identity values between strain 13AT and each of the reference type strains were 72.5, 72.9, 72.3, 75.0 and 69.2 %, respectively. The major fatty acids were iso-C15 : 0, iso-C16 : 0 and summed feature 9 (iso-C17 : 1 ω9c and/or C16 : 0 10-methyl). The sole respiratory quinone was identified as ubiquinone-8. The polar lipid profile contained phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, an unidentified aminolipid, an unidentified lipid, four unidentified phospholipids and two unidentified glycolipids. Based on the phenotypic, physiological, phylogenetic and chemotaxonomic data, strain 13AT represents a novel species of the genus Lysobacter, for which the name Lysobacter selenitireducens sp. nov. is proposed. The type strain is 13AT (=JCM 34786T=GDMCC 1.2722T).

Parvicella tangerina gen. nov., sp. nov. (Parvicellaceae fam. nov., Flavobacteriales), first cultured representative of the marine clade UBA10066, and Lysobacter luteus sp. nov., from activated sludge of a seawater-processing wastewater treatment plant.

Two strains isolated from a sample of activated sludge that was obtained from a seawater-based wastewater treatment plant on the southeastern Mediterranean coast of Spain have been characterized to achieve their taxonomic classification, since preliminary data suggested they could represent novel taxa. Given the uniqueness of this habitat, as this sort of plants are rare in the world and this one used seawater to process an influent containing intermediate products from amoxicillin synthesis, we also explored their ecology and the annotations of their genomic sequences. Analysis of their 16S rRNA gene sequences revealed that one of them, which was orange-pigmented, was distantly related to Vicingus serpentipes (family Vicingaceae) and to other representatives of neighbouring families in the order Flavobacteriales (class Flavobacteriia) by 88-89 % similarities; while the other strain, which was yellow-pigmented, was a putative new species of Lysobacter (family Xanthomonadaceae, order Xanthomonadales, class Gammaproteobacteria) with Lysobacter arseniciresistens as closest relative (97.3 % 16S rRNA sequence similarity to its type strain). Following a polyphasic taxonomic approach, including a genome-based phylogenetic analysis and a thorough phenotypic characterization, we propose the following novel taxa: Parvicella tangerina gen. nov., sp. nov. (whose type strain is AS29M-1T=CECT 30217T=LMG 32344T), Parvicellaceae fam. nov. (whose type genus is Parvicella), and Lysobacter luteus sp. nov. (whose type strain is AS29MT=CECT 30171T=LMG 32343T).

Biosynthesis of Odd-Carbon Unsaturated Fatty Dicarboxylic Acids Through Engineering the HSAF Biosynthetic Gene in Lysobacter enzymogenes.

Fatty dicarboxylic acids (FDCA) are useful as starting materials or components for plastics, polyesters, nylons, and fragrances. Most of the commercially available FDCA contain an even number of carbons, and there remain few sustainable methods for production of FDCA with an odd number of carbons (o-FDCA). In this work, we explored a novel biosynthetic route to unsaturated o-FDCA. The approach was based on genetic modifications of hsaf pks-nrps, encoding a hybrid polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) in Lysobacter enzymogenes, an environmental bacterium emerging as a new biocontrol agent. This single-module PKS-NRPS catalyzes the biosynthesis of lysobacterene A, a polyene-containing precursor of the antifungal natural product Heat-Stable Antifungal Factor (HSAF). We genetically removed the NRPS module from this gene and generated a new strain of L. enzymogenes, in which the PKS module was fused to the thioesterase domain of hsaf pks-nrps. The chimeric gene was verified by DNA sequencing, and its expression in L. enzymogenes was confirmed by reverse transcription-polymerase chain reaction (RT-PCR). The total fatty acids were extracted, esterified, and analyzed by GC-MS. The results showed that the engineered strain produced new fatty acids that were absent in the wild type. The main product was identified as hepta-2,4-dienedioic acid, an unsaturated o-FDCA. This work sets the foundation to explore a sustainable and environment-friendly approach toward unsaturated o-FDCA, which could be used as precursors for new compounds that can serve as versatile feedstock for industrial materials.

Lysobacter chinensis sp. nov., a cellulose-degrading strain isolated from cow dung compost.

A novel bacterial strain, TLK-CK17T, was isolated from cow dung compost sample. The strain was Gram-staining negative, non-gliding rods, aerobic, and displayed growth at 15-40 °C (optimally, 35 °C), with 0-5.0% (w/v) NaCl (optimally, 0.5) and at pH 6.5-8.5 (optimally, 7.0-7.5). The assembled genome of strain TLK-CK17T has a total length of 4.3 Mb with a G + C content of 68.2%. According to the genome analysis, strain TLK-CK17T encodes quite a few glycoside hydrolases that may play a role in the degradation of accumulated plant biomass in compost. On the basis 16S rRNA gene sequence analysis, strain TLK-CK17T showed the highest sequence similarity (98.9%) with L. penaei GDMCC 1.1817 T, followed by L. maris KCTC 42381 T (98.3%). Cells contained iso-C16:0, iso-C15:0, and summed feature 9 (comprising C17:1 ω9c and/or 10-methyl C16:0), as its major cellular fatty acids (> 10.0%) and ubiquinone-8 as the exclusively respiratory quinone. Diphosphatidylglycerol, phosphatidylethanolamine, and phosphatidylglycerol prevailed among phospholipids. Based on the phenotypic, genomic and phylogenetic data, strain TLK-CK17T represents a novel species of the genus Lysobacter, for which the name Lysobacter chinensis sp. nov. is proposed, and the type strain is TLK-CK17T (= CCTCC AB2021257T = KCTC 92122 T).

The First Homologous Expression System for the ß-Lytic Protease of Lysobacter capsici VKM B-2533T, a Promising Antimicrobial Agent.

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, ß-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.

Enhancing proteolytic activity of Lysobacter enzymogenes using cold atmospheric plasma.

Cold atmospheric plasma (CAP) is being used recently as a modern technique for microbial random mutagenesis. In the present study, CAP was used to induce mutagenesis in L. enzymogenes which is the bacteria known for producing proteolytic enzymes especially lysyl endopeptidase (Lys C). Enhanced proteolytic activity was the main criteria to select mutant strains. Therefore, the cell suspension of L. enzymogenes strain (ATCC 29487), was exposed to CAP for 30, 45, 90, and 150 s. The proteolytic activity of mutant strains was screened initially by radial caseinolytic assay and then by Ansons method in different phases of bacterial growth in the selected mutants. The purification process of Lysyl endopeptidase as the target enzyme was optimized and for enlightening molecular aspect of CAP mutagenesis, the sequences of the upstream and coding regions of lys C gene from 10 selected mutant strains were determined. The bacterial survival assessment showed that the more CAP treatment time, the less survival rate, however, in all exposure times, a number of survived mutants showed enhanced proteolytic activity. Among 38 out of 100 examined mutants which showed higher proteolytic activity than that of wild type, the M1-30 s mutant exhibited the highest increment to 1.94 fold. The SDS-PAGE analysis showed expected size of purified Lys C from M1-30 s. The Lys C gene from M14-150 s mutant strain (1.4-fold increment) harbored three point mutations which can be effective in enhancing protease activity. In conclusion, the results highlighted the role of CAP for strain improvement process to obtain industrial strains.

Lysobacter ciconiae sp. nov., and Lysobacter avium sp. nov., isolated from the faeces of an Oriental stork.

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.

Exploiting the antibacterial mechanism of phenazine substances from Lysobacter antibioticus 13-6 against Xanthomonas oryzae pv. oryzicola.

Bacterial leaf streak caused by Xanthomonas oryzae pv. oryzicola (Xoc) is one of the most destructive diseases affecting rice production worldwide. In this study, we extracted and purified phenazine substances from the secondary metabolites of Lysobacter antibioticus 13-6. The bacteriostatic mechanism of phenazine substances against Xoc was investigated through physiological response and transcriptomic analysis. Results showed that phenazine substances affects the cell membrane permeability of Xoc, which causes cell swelling and deformation, blockage of flagellum synthesis, and imbalance of intracellular environment. The changes in intracellular environment affect the physiological and metabolic functions of Xoc, which reduces the formation of pathogenic factors and pathogenicity. Through transcriptomic analysis, we found that among differentially expressed genes, the expression of 595 genes was induced significantly (275 up-regulated and 320 down-regulated). In addition, we observed that phenazine substances affects three main functions of Xoc, i.e., transmembrane transporter activity, DNA-mediated transposition, and structural molecular activity. Phenazine substances also inhibits the potassium ion transport system that reduces Xoc resistance and induces the phosphate ion transport system to maintain the stability of the internal environment. Finally, we conclude that phenazine substances could retard cell growth and reduce the pathogenicity of Xoc by affecting cell structure and physiological metabolism. Altogether, our study highlights latest insights into the antibacterial mechanism of phenazine substances against Xoc and provides basic guidance to manage the incidence of bacterial leaf streak of rice.