Gymnodinialimonas phycosphaerae sp. nov., a phycosphere bacterium isolated from Karlodinium veneficum.

A facultative anaerobic, Gram-negative, non-motile, rod-shaped bacterial strain, designated N5T, was obtained from the phycosphere microbiota of the marine planktonic dinoflagellate, Karlodinium veneficum. Strain N5T showed growth on marine agar at 25 °C, pH 7 and 1 % (w/v) NaCl and produced a yellow colour. According to a phylogenetic study based on 16S rRNA gene sequences, strain N5T has a lineage within the genus Gymnodinialimonas. The G+C content in the genome of strain N5T is 62.9 mol% with a total length of 4 324 088 bp. The NCBI Prokaryotic Genome Annotation Pipeline revealed that the N5T genome contained 4230 protein-coding genes and 48 RNA genes, including a 5S rRNA, 16S rRNA, 23S rRNA, 42 tRNA, and three ncRNAs. Genome-based calculations (genome-to-genome distance, average nucleotide identity and DNA G+C content) clearly indicated that the isolate represents a novel species within the genus Gymnodinialimonas. The predominant fatty acids were C19 : 0 cyclo ω8c and feature 8 (comprising C18 : 1 ω6c and/or C18 : 1 ω7c). The major polar lipids were phosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine. The main respiratory quinone was Q-10. Based on its phenotypic, phylogenetic, genomic and chemotaxonomic features, strain N5T represents a novel species of the genus Gymnodinialimonas, for which the name Gymnodinialimonas phycosphaerae sp. nov. is proposed. The type strain is N5T (=KCTC 82362T=NBRC 114899T).

A nostoxanthin-producing bacterium, Sphingomonas nostoxanthinifaciens sp. nov., alleviates the salt stress of Arabidopsis seedlings by scavenging of reactive oxygen species.

A novel, nostoxanthin-producing, endophytic bacterium, designated as AK-PDB1-5T, was isolated from the needle-like leaves of the Korean fir (Abies koreana Wilson) collected from Mt. Halla in Jeju, South Korea. A 16S rRNA sequence comparison indicated that the closest phylogenetic neighbors were Sphingomonas crusticola MIMD3T (95.6%) and Sphingomonas jatrophae S5-249T (95.3%) of the family Sphingomonadaceae. Strain AK-PDB1-5T had a genome size of 4,298,284 bp with a 67.8% G + C content, and digital DNA-DNA hybridization and OrthoANI values with the most closely related species of only 19.5-21% and 75.1-76.8%, respectively. Cells of the strain AK-PDB1-5T were Gram-negative, short rods, oxidase- and catalase-positive. Growth occurred at pH 5.0-9.0 (optimum pH 8.0) in the absence of NaCl at 4-37°C (optimum 25-30°C). Strain AK-PDB1-5T contained C14:0 2OH, C16:0 and summed feature 8 as the major cellular fatty acids (> 10%), while sphingoglycolipid, phosphatidylethanolamine, phosphatidylglycerol, phospholipids and lipids were found to be the major polar lipids. The strain produces a yellow carotenoid pigment; natural products prediction via AntiSMASH tool found zeaxanthin biosynthesis clusters in the entire genome. Biophysical characterization by ultraviolet-visible absorption spectroscopy and ESI-MS studies confirmed the yellow pigment was nostoxanthin. In addition, strain AK-PDB1-5T was found significantly promote Arabidopsis seedling growth under salt conditions by reducing reactive oxygen species (ROS). Based on the polyphasic taxonomic analysis results, strain AK-PDB1-5T was determined to be a novel species in the genus Sphingomonas with the proposed name Sphingomonas nostoxanthinifaciens sp. nov. The type strain is AK-PDB1-5T (= KCTC 82822T = CCTCC AB 2021150T).

Genome insights into the plant growth-promoting bacterium Saccharibacillus brassicae ATSA2T.

Endophytes can facilitate the improvement of plant growth and health in agriculturally important crops, yet their genomes and secondary metabolites remain largely unexplored. We previously isolated Saccharibacillus brassicae strain ATSA2T from surface-sterilized seeds of kimchi cabbage and represented a novel species of the genus Saccharibacillus. In this study, we evaluated the plant growth-promoting (PGP) effect of strain ATSA2T in kimchi cabbage, bok choy, and pepper plants grown in soils. We found a significant effect on the shoot and root biomass, and chlorophyll contents following strain ATSA2T treatment. Strain ATSA2T displayed PGP traits such as indole acetic acid (IAA, 62.9 µg/mL) and siderophore production, and phosphate solubilization activity. Furthermore, genome analysis of this strain suggested the presence of gene clusters involved in iron acquisition (fhuABD, afuABC, fbpABC, and fepCDG) and phosphate solubilization (pstABCHS, phoABHLU, and phnCDEP) and other phytohormone biosynthesis genes, including indole-3-acetic acid (trpABCDEFG), in the genome. Interestingly, the secondary metabolites cerecidin, carotenoid, siderophore (staphylobactin), and bacillaene underlying plant growth promotion were found in the whole genome via antiSMASH analysis. Overall, physiological testing and genome analysis data provide comprehensive insights into plant growth-promoting mechanisms, suggesting the relevance of strain ATSA2T in agricultural biotechnology.

Sphingomonas cannabina sp. nov., isolated from Cannabis sativa L. 'Cheungsam'.

A Gram-negative, aerobic, rod-shaped bacterium, designated DM2-R-LB4T was isolated from Cannabis sativa L. 'Cheungsam' in Andong, Republic of Korea. The strain DM2-R-LB4T grew at temperatures of 15-45 °C (optimum, 30-37 °C), pH of 5.5-9 (optimum, 8.0), and 0-2 % (w/v) NaCl concentration (optimum, 0%). Phylogenetic analyses based on the 16S rRNA gene sequences revealed that strain DM2-R-LB4T is related to species of the genus Sphingomonas, and shared 97.8 and 97.5% similarity to Sphingomonas kyenggiensis KCTC 42244T and Sphingomonas leidyi DSM 4733T, respectively. The DNA G+C content was 67.9 mol% and genome analysis of the strain DM2-R-LB4T revealed that the genome size was 4 386 171 bp and contained 4 009 predicted protein-coding genes. The average nucleotide identity (ANI) values between strain DM2-R-LB4T and S. kyenggiensis KCTC 42244T, and S. leidyi DSM 4733T was 76.8 and 76.7 %, respectively, while the values of digital DNA-DNA hybridization (dDDH) were 20.7 and 20.6 %, respectively. C14 : 0 2-OH, C16 : 0, and summed feature 8 (C18 : 1 ω6c and/or C18 : 1 ω7c) were the major fatty acids (>10 %) in the strain DM2-R-LB4T. The polar lipids comprised diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylcholine (PC), sphingoglycolipid (SGL), glycolipid (GL), phospholipid (PL), and two unidentified polar lipids (L1 and L2). Ubiquinone-10 (Q-10) was the only respiratory quinone. The polyamine pattern was found to contain homospermidine, putrescine, and spermidine. The results of phylogenetic anlayses, polyphasic studies, revealed that strain DM2-R-LB4T represents a novel species of the genus Sphingomonas, for which the name Sphingomonas cannabina sp. nov., is proposed. The type strain is DM2-R-LB4T (=KCTC 92075T = GDMCC 1.3018T).

Subtercola endophyticus sp. nov., a cold-adapted bacterium isolated from Abies koreana.

A novel Gram-stain-positive, aerobic bacterial strain, designated AK-R2A1-2 T, was isolated from the surface-sterilized needle leaves of an Abies koreana tree. Strain AK-R2A1-2 T had 97.3% and 96.7% 16S rRNA gene sequence similarities with Subtercola boreus K300T and Subtercola lobariae 9583bT, respectively, but formed a distinct phyletic lineage from these two strains. Growth of strain AK-R2A1-2 T was observed at 4-25 °C at pH 5.0-8.0. Strain AK-R2A1-2 T contained menaquinone 9 (MK-9) and menaquinone 10 (MK-10) as the predominant respiratory quinones. The major cellular fatty acids were anteiso-C15:0 and summed feature 8 (C18:1ω7c or/and C18:1ω6c), and the polar lipids included diphosphatidylglycerol (DPG) and three unknown aminolipids, AKL2, AKL3, and AKL4. The complete genome of strain AK-R2A1-2 T was sequenced to understand the genetic basis of its survival at low temperatures. Multiple copies of cold-associated genes involved in cold-active chaperon, stress response, and DNA repair supported survival of the strain at low temperatures. Strain AK-R2A1-2 T was also able to significantly improve rice seedling growth under low temperatures. Thus, this strain represents a novel species of the genus Subtercola, and the proposed name is Subtercola endophyticus sp. nov. The type strain is AK-R2A1-2 T (= KCTC 49721 T = GDMCC 1.2921 T).

Flavobacterium endoglycinae sp. nov., an endophytic bacterium isolated from soybean (Glycine max L. cv. Gwangan) stems.

A Gram-stain-negative, facultatively anaerobic, motile by gliding, rod-shaped, oxidase- and catalase-positive bacterial strain, designated BB8T, was isolated from the stems of a Korean soybean cultivar (Glycine max L. cv. Gwangan). The strain produced a yellow pigment on tryptic soy agar. Growth of strain BB8T occurred at pH 5.0-8.0 (optimum, pH 7.0), at 10-35 °C (optimum, 25-30 °C) and in the presence of 0-1 % (w/v) NaCl (optimum, 0.5%). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain BB8T formed a lineage within the genus Flavobacterium and was most closely related to Flavobacterium artemisiae SYP-B1015T (96.9 % 16S rRNA gene sequence similarity) and Flavobacterium ustbae T13T (96.8%). The complete genome sequence of strain BB8T was 5 513 159 bp long with a G+C content of 34.1 mol%. The major fatty acids (>10 %) of strain BB8T were iso-C15 : 0 (21 %), summed feature 3 (comprising C16 : 1 ω7c and/or C16 : 1 ω6c, 20.3%) and iso-C16 : 0 3-OH (13.7%). The predominant polar lipids were phosphatidylethanolamine and unidentified aminolipids, and the major respiratory quinone was menaquinone-6. Based on these phenotypic, genotypic and chemotaxonomic characteristics, strain BB8T is considered to represent a novel species of the genus Flavobacterium, for which the name Flavobacterium endoglycinae sp. nov. is proposed. The type strain is BB8T (=KCTC 82167T=CCTCC AB 2020070T).

Pyomelanin-Producing Brevundimonas vitisensis sp. nov., Isolated From Grape (Vitis vinifera L.).

A novel endophytic bacterial strain, designated GR-TSA-9T, was isolated from surface-sterilized grape (Vitis vinifera L.). 16S rRNA gene sequence analyses showed that the isolate was grouped within the genus Brevundimonas, displaying the highest similarity with Brevundimonas lenta DS-18T (97.9%) and Brevundimonas kwangchunensis KSL-102T (97.8%) and less than 97.5% similarity with other members of Brevundimonas. The strain GR-TSA-9T was a gram negative, rod shaped, facultatively anaerobic, catalase and oxidase positive, and motile bacterium. Its growth occurred at 10-37°C (optimally 25-30°C), at pH 7.0-8.0, and in NaCl 0-1% (optimally 0%). It contained ubiquinone-10 as a respiratory quinone, and the major cellular fatty acids (>10% of the total) were C16:0 (14.2%) and summed feature 8 (C18:1ω7c and/or C18:1ω6c, 65.6%). The polar lipids present in the strain were phosphoglycolipids, phosphatidylglycerol, 1,2-di-O-acyl-3-O-[d-glucopyranosyl-(1→4)-α-d-glucopyranuronosyl]glycerol, and unidentified lipids (L1, L2, and L4). The strain had one 2,976,716bp circular chromosome with a G+C content of 66.4%. The digital DNA-DNA hybridization value between strain GR-TSA-9T and B. lenta DS-18T was 20.9%, while the average nucleotide identity value was 76.7%. In addition, the dDDH and ANI values to other members in this genus, whose genome sequences are available, are less than 21.1 and 77.6%. Genome annotation predicted the presence of some gene clusters related to tyrosine degradation and pyomelanin formation. Strain GR-TSA-9T produced a brown melanin-like pigment in the presence of L-tyrosine-containing media. The highest pigment production (0.19g/L) was observed in tryptic soy broth with 1.0mg/ml L-tyrosine at 25°C for 6days of culture. Biophysical characterization by ultraviolet (UV)-visible spectroscopy, Fourier-transform infrared spectroscopy, and electrospray ionization mass spectrometry confirmed that the pigment was pyomelanin. Additionally, melanized GR-TSA-9T cells could protect the cells against UVC exposure. The phylogenetic, genomic, phenotypic, and chemotaxonomic features indicated that strain GR-TSA-9T represents a novel melanin-producing species of Brevundimonas. The type strain was GR-TSA-9T (KCTC 82386T=CGMCC 1.18820T).

Pedobacter endophyticus sp. nov., an endophytic bacterium isolated from Carex pumila.

An aerobic, Gram-stain-negative, weak-motile, short-rod-shaped bacterial strain, designated JBR3-12T, was isolated from halophyte Carex pumila plants, and its taxonomic position was investigated by using a polyphasic taxonomic approach. The strain produced a pink pigment on tryptic soy agar and grew optimally at 25 °C, pH 8 and in the presence of 3 % (w/v) NaCl. Results of phylogenetic analysis based on 16S rRNA gene sequences showed that strain JBR3-12T formed a lineage within the genus Pedobacter and was most closely related to Pedobacter sandarakinus DS-27T (98.0 %) and Pedobacter agri PB92T (97.6 %). The DNA G+C content of the genome was 41.3 mol%; the whole genome length was 5 426 070 bp. The major fatty acids of JBR3-12T were iso-C15 : 0, summed feature 3 (comprising C16 : 1 ω6c and/or C16 : 1 ω7c) and iso-C17 : 0 3-OH. The predominant polar lipid was phosphatidylethanolamine. The predominant quinone was menaquinone-7. Based on its phenotypic, phylogenetic and genotypic features, strain JBR3-12T is proposed to represent a novel species of the genus Pedobacter, for which the name is Pedobacter endophyticus sp. nov. The type strain is JBR3-12T (=KCTC 82363T=NBRC 114901T).

Artificial oxidative stress-tolerant Corynebacterium glutamicum.

We have reported a transcription profile of an adapted Corynebacterium glutamicum that showed enhanced oxidative stress resistance. To construct an artificial oxidative stress-resistant strain, gene clusters in the ß-ketoadipate pathway, which were up-regulated in the adapted strain, were artificially expressed in the wild-type C. glutamicum. The wild-type strain was unable to grow under 2 mM H2O2 containing minimal medium, while the strains expressing pca gene clusters restored growth under the same medium, and the pcaHGBC expression showed the most significant effect among the gene clusters. The expressions of pca gene clusters also enabled the wild-type to increase its resistance against oxidative stressors, such as diamide and cumene hydroperoxide, as well as H2O2. The oxidative stress tolerance of the strain was correlated to the reactive oxygen species (ROS)-scavenging activity of the cell extract. The reason for the enhanced oxidative stress-resistance of C. glutamicum and its applications on the synthetic strain development are discussed.

Estimation of phosphoenolpyruvate carboxylation mediated by phosphoenolpyruvate carboxykinase (PCK) in engineered Escherichia coli having high ATP.

We have previously reported that phosphoenolpyruvate carboxykinase (PCK) overexpression under glycolytic conditions enables Escherichia coli to harbor a high intracellular ATP pool resulting in enhanced recombinant protein synthesis. To estimate how much PCK-mediated phosphoenolpyruvate (PEP) carboxylation is contributed to the ATP increase under engineered conditions, the kinetics of PEP carboxylation by PCK and substrate competing phosphoenolpyruvate carboxylase (PPC) were measured using recombinant enzymes. The PEP carboxylation catalytic efficiency (kcat/Km) of the recombinant PCK was 660mM(-1)min(-1), whereas that of the recombinant PPC was 1500mM(-1)min(-1). Under the presence of known allosteric effectors (fructose 1,6-bisphosphate, acetyl-CoA, ATP, malate, and aspartate) close to in vivo conditions, the catalytic efficiency of PCK-mediated PEP carboxylation (84mM(-1)min(-1)) was 28-folds lower than that of PPC (2370mM(-1)min(-1)). To verify the above results, an E. coli strain expressing native PCK and PPC under control of identical promoter was constructed by replacing PCK promoter region with that of PPC in chromosome. The native PCK activity (33nmol/mg-proteinmin) was 5-folds lower than PPC activity (160nmol/mg-proteinmin) in the cell extract from the promoter-exchanged strain. Intracellular modifications of ATP concentration by PCK activity and the consequences for biotechnology are further discussed.

Adaptive evolution of Corynebacterium glutamicum resistant to oxidative stress and its global gene expression profiling.

Corynebacterium glutamicum was adapted in a chemostat for 1,900 h with gradually increasing H2O2 stress to understand the oxidative stress response of an industrial host. After 411 generations of adaptation, C. glutamicum developed the ability to grow under stress of 10 mM H2O2, whereas the wild-type did not. The adapted strain also showed increased stress resistance to diamide and menadione, SDS, Tween 20, HCl, NaOH, and ampicillin. A total of 1,180 genes in the RNA-seq transcriptome analysis of the adapted strain were up-regulated twice or higher (corresponding to 38.6 % of the genome), and 126 genes were down-regulated half or less (4.1 % of genome) under 10 mM H2O2-stress conditions compared with those of the wild-type under a no stress condition. Especially the aromatic compound-degrading gene clusters (vanRABK, pcaJIRFLO, and benABCDRKE) were more than threefold up-regulated. Plausible reasons for the H2O2-stress tolerance of the adapted strain are discussed as well as the potential strategy for development of oxidative stress-tolerant strain.

Analysis and functional expression of NPP pathway-specific regulatory genes in Pseudonocardia autotrophica.

Using the genomics-guided polyene screening method, a rare actinomycetes called Pseudonocardia autotrophica was previously identified to contain functionally clustered nystatin-like biosynthetic genes and to produce a presumably novel polyene compound named nystatin-like Pseudonocardia polyene (NPP) (Kim et al., J Ind Microbiol Biotechnol 36:1425-1434, 2009). Since very low NPP productivity was observed in most P. autotrophica culture conditions, its biosynthetic pathway was proposed to be tightly regulated. Herein we report in silico analysis of six putative NPP pathway-specific regulatory genes present in its biosynthetic gene cluster, followed by functional overexpression of these regulatory genes in P. autotrophica. Three pathway-specific regulatory genes (nppRI, RIII, and RV) were predicted to belong to a typical LAL-type transcriptional family. Each regulatory gene was cloned under the strong constitutive ermE* promoter in a Streptomyces integrative pSET152 plasmid, followed by direct intergeneric conjugation from a plasmid-containing E. coli donor cell to P. autotrophica. While all the P. autotrophica exconjugants exhibited improved NPP productivity, the one containing nppRIII showed the highest NPP productivity improvement. In addition, culture-time-dependent analysis revealed that the nppRIII-stimulated NPP biosynthesis was more significant in the late exponential growth stage than in the stationary stage. Moreover, the P. autotrophica nppRIII-disruption mutant failed to produce NPP, with significantly reduced transcription levels of most npp biosynthetic genes. The results described suggest that identification and overexpression of key pathway-specific regulatory gene, followed by optimum harvest timing, should be critical factors to maximize the productivity of an intrinsically low-level metabolite such as NPP produced by rare actinomycetes species.

Identification of functionally clustered nystatin-like biosynthetic genes in a rare actinomycetes, Pseudonocardia autotrophica.

The polyene antibiotics, including nystatin, pimaricin, amphotericin, and candicidin, comprise a family of very valuable antifungal polyketide compounds, and they are typically produced by soil actinomycetes. Previously, using a polyene cytochrome P450 hydroxylase-specific genome screening strategy, Pseudonocardia autotrophica KCTC9441 was determined to contain genes potentially encoding polyene biosynthesis. Here, sequence information of an approximately 125.7-kb contiguous DNA region in five overlapping cosmids isolated from the P. autotrophica KCTC9441 genomic library revealed a total of 23 open reading frames, which are presumably involved in the biosynthesis of a nystatin-like compound tentatively named NPP. The deduced roles for six multi-modular polyketide synthase (PKS) catalytic domains were found to be highly homologous to those of previously identified nystatin biosynthetic genes. Low NPP productivity suggests that the functionally clustered NPP biosynthetic pathway genes are tightly regulated in P. autotrophica. Disruption of a NPP PKS gene completely abolished both NPP biosynthesis and antifungal activity against Candida albicans, suggesting that polyene-specific genome screening may constitute an efficient method for isolation of potentially valuable previously identified polyene genes and compounds from various rare actinomycetes widespread in nature.