Sphingomonas Aquatilis Research Articles

Anti-Oomycete Activity and Pepper Root Colonization of Pseudomonas plecoglossicida YJR13 and Pseudomonas putida YJR92 against Phytophthora capsici

Previously, Pseudomonas plecoglossicida YJR13 and Pseudomonas putida YJR92 from a sequential screening procedure were proven to effectively control Phytophthora blight caused by Phytophthora capsici. In this study, we further investigated the anti-oomycete activities of these strains against mycelial growth, zoospore germination, and germ tube elongation of P. capsici. We also investigated root colonization ability of the bacterial strains in square dishes, including cell motility (swimming and swarming motilities) and biofilm formation. Both strains significantly inhibited mycelial growth in liquid and solid V8 juice media and M9 minimal media, zoospore germination, and germ tube elongation compared with Bacillus vallismortis EXTN-1 (positive biocontrol strain), Sphingomonas aquatilis KU408 (negative biocontrol strain), and MgSO4 solution (untreated control). In diluted (nutrient-deficient) V8 juice broth, the tested strain populations were maintained at >108 cells/ml, simultaneously providing mycelial inhibitory activity. Additionally, these strains colonized pepper roots at a 106 cells/ml concentration for 7 days. The root colonization of the strains was supported by strong swimming and swarming activities, biofilm formation, and chemotactic activity towards exudate components (amino acids, organic acids, and sugars) of pepper roots. Collectively, these results suggest that strains YJR13 and YJR92 can effectively suppress Phytophthora blight of pepper through direct anti-oomycete activities against mycelial growth, zoospore germination and germ tube elongation. Bacterial colonization of pepper roots may be mediated by cell motility and biofilm formation together with chemotaxis to root exudates.

Kombucha tea beverage: Microbiological characteristic, antioxidant activity, and phytochemical composition

The aim of the present study was to determine the microbial composition, antioxidant activity, and content of phytochemicals in prepared kombucha tea beverage. Microbiota was identified by MALDI-TOF mass spectrometry, antioxidant activity of beverage was tested by ABTS and phosphomolybdenum method, the total content of phytochemicals (polyphenols, flavonoids, and phenolic acids) was measured by colorimetric methods. The major phenolic acids, flavonoids, and methylxanthines were detected by high performance liquid chromatography (HPLC). Candida krusei, Sphingomonas melonis, Sphingomonas aquatilis, Brevibacillus centrosporus, and Gluconobacter oxydans were the most abundant microorganisms. Antioxidant activity of kombucha tested by ABTS and phosphomolybdenum method was 1.16 mg TEAC/ml and 2.04 mg TEAC/ml, respectively, which values were higher than in black tea 0.67 and 0.81 mg TEAC/ml, respectively. Also, content of total polyphenols (0.42 mg GAE/ ml), flavonoids (0.13 mg QE/ml), and phenolic acids (0.19 mg CAE/ml) was higher in kombucha than in black tea (0.18 mg GAE/ml; 0.02 mg QE/ml; 0.05 mg CAE/ml, respectively). Gallic, chlorogenic, syringic, and protocatechuic acids, and rutin and vitexin from flavonoids were dominant in kombucha beverage detected by HPLC. Strong difference in caffeine contents, 217.81 µg ml−1 (black tea) and 100.72 µg ml−1 (kombucha beverage), was observed. The amounts of theobromine were similar in black tea and kombucha, but theophylline was detected only in black tea in trace amount (0.52 µg ml−1).

Occurrence of diversified N‐acyl homoserine lactone mediated biofilm‐forming bacteria in rice rhizoplane

Quorum sensing (QS)-mediated biofilm-forming rhizobacteria are indispensable due to their competitiveness in the crop rhizosphere. In the present work, we have reported on the occurrence of diversified bacterial species capable of producing N-acyl homoserine lactone (AHL) as the QS signal in the roots of arice plant grown under field conditions. The AHL-producing bacteria were directly isolated from the rice root by the biosensor reporter (Chromobacterium violaceum CV026) overlay method and characterized for biofilm production by the microtiter plate method. A total of 48 QS-positive bacterial isolates were purified from different aged (7, 20, 24, 26, and 36 days) rice seedlings. The in vitro biofilm production and genetic diversity as revealed by BOX-PCR fingerprinting showed high variability among the isolates. Most of the best biofilm-forming isolates produced aN-butyryl dl-homoserine lactone (a C4-AHL type) signal in the medium. The 16S ribosomal RNA (rRNA) gene sequence of these putative elite isolates identified that they were close to Aeromonas hydrophila (QS7-4; QS36-2), A. enteropelongenes (QS20-8), A. veronii (QS36-3), Enterobacter sp. (QS20-11), Klebsiella pneumoniae (QS24-6), Kosakonia cowanii (QS24-21), Providentia rettigeri (QS24-2), Sphingomonas aquatilis (QS24-17), and Pseudomonas sihuiensis (QS24-20). These strains profusely colonized the rice root upon inoculation and formed biofilms on the surface of the root under gnotobiotic conditions. Developing inoculants from these strains would ensure competitive colonization on the rhizoplane of the crop through their biofilm and thereby improve plant growth and health.

Biocontrol Activity of Volatile-Producing Bacillus megaterium and Pseudomonas protegens against Aspergillus flavus and Aflatoxin Production on Stored Rice Grains

In our previous study, three bacterial strains, Bacillus megaterium KU143, Microbacterium testaceum KU313, and Pseudomonas protegens AS15, were selected as effective biocontrol agents against Aspergillus flavus on stored rice grains. In this study, we evaluated the inhibitory effects of the volatiles produced by the strains on A. flavus growth and aflatoxin production on stored rice grains. The three strains significantly reduced mycelial growth of A. flavus in dual-culture assays compared with the negative control strain, Sphingomonas aquatilis KU408, and an untreated control. Of these tested strains, volatiles produced by B. megaterium KU143 and P. protegens AS15 markedly inhibited mycelial growth, sporulation, and conidial germination of A. flavus on agar medium and suppressed the fungal populations in rice grains. Moreover, volatiles produced by these two strains significantly reduced aflatoxin production in the rice grains by A. flavus. To our knowledge, this is the first report of the suppression of A. flavus aflatoxin production in rice grains using B. megaterium and P. protegens volatiles.

Biocatalytic Asymmetric Synthesis of (1R, 2S)-N-Boc-vinyl-ACCA Ethyl Ester with a Newly Isolated Sphingomonas aquatilis.

1-amino cyclopropane-1-carboxylic acid (ACCA) and its derivatives are essential pharmacophoric unit that widely used in drug research and development. Specifically, (1R, 2S)-N-Boc-vinyl-ACCA ethyl ester (vinyl-ACCA) is a key chiral intermediate in the synthesis of highly potent hepatitis C virus (HCV) NS3/4A protease inhibitors such as asunaprevir and simeprevir. Developing strategies for the asymmetric synthesis of vinyl-ACCA is thus extremely high demand. In this study, 378 bacterial strains were isolated from soil samples using N-Boc-vinyl-ACCA ethyl ester as the sole carbon source and were screened for esterase activity. Fourteen of which worked effectively for the asymmetric synthesis of (1R, 2S)-N-Boc-1-vinyl ACCA ethyl ester. The strain CY-2, identified as Sphingomonas aquatilis, which showed the highest stability and enantioselectivity was selected as whole cell biocatalyst for further study. A systematic study of all factors influencing the enzymatic hydrolysis was performed. Under optimized conditions, resolution of rac-vinyl-ACCA to (1R, 2S)-N-Boc-1-vinyl ACCA ethyl ester with 88.2% ee and 62.4% conversion (E=9) was achieved. Besides, S. aquatilis was also used to transform other 10 different substrates. Notably, it was found that 7 of them could be stereoselectively hydrolyzed, especially for (1R,2S)-1-amino-vinyl-ACCA ethyl ester hydrochloride (99.6% ee, E>200). Our investigations provide a new efficient whole cell biocatalyst for resolution of ACCA and might be developed for industry application.

Two Novel Sets of Genes Essential for Nicotine Degradation by Sphingomonas melonis TY.

Nicotine is a type of environmental pollutant present in the tobacco waste that is generated during tobacco manufacturing. Sphingomonas melonis TY can utilize nicotine as a sole source of carbon, nitrogen and energy via a variant of the pyridine and pyrrolidine pathway (the VPP pathway). In this study, we report the identification of two novel sets of genes, ndrA1A2A3, and ndrB1B2B3B4, which are crucial for nicotine degradation by strain TY. ndrA1A2A3 and ndrB1B2B3B4 exhibit similarity with both nicotine dehydrogenase ndh from Arthrobacter nicotinovorans and nicotine hydroxylase vppA from Ochrobactrum sp. SJY1. The transcriptional levels of ndrA1A2A3 and ndrB1B2B3B4 in strain TY were significantly upregulated in the presence of nicotine. Furthermore, ndrA1 or ndrB2 knockout resulted in a loss of the ability to degrade nicotine, whereas gene complementation restored the capacity of each mutant to utilize nicotine for growth. Biodegradation assays indicated that the mutant strains retained the ability to degrade the first intermediate in the pathway, 6-hydroxynicotine (6 HN). However, heterologous expression of ndrA1A2A3 and ndrB1B2B3B4 did not confer nicotine dehydrogenase activity to E. coli DH5α, Pseudomonas putida KT2440 or Sphingomonas aquatilis. These results provide information on the VPP pathway of nicotine degradation in S. melonis TY, and we conclude that these two sets of genes have essential functions in the conversion of nicotine to 6 HN in strain TY.

Sphingomonas rubra sp. nov., isolated from bioreactor wastewater

A Gram-reaction-negative, rod-shaped, motile, neutrophilic bacterium, designated strain BH3(T), was isolated from wastewater of a sequential batch reactor treating wastewater taken from a leather plant. The isolate grew in 0-8 % (w/v) NaCl, at pH 6-9 and at 4-45 °C. Chemotaxonomic analysis showed that strain BH3(T) had characteristics typical of members of the genus Sphingomonas, such as the presence of sphingolipids, Q-10 and 2-hydroxymyristic acid and the absence of 3-hydroxy fatty acids. The presence of C(18 : 1)ω7c (39.2 %) and C(16 : 0) (11.2 %) as major fatty acids, C(14 : 0) 2-OH (20.6 %) as the major 2-hydroxy fatty acid and homospermidine as the major polyamine indicated that strain BH3(T) belonged to the genus Sphingomonas sensu stricto. The genomic DNA G+C content of strain BH3(T) was 65.6 mol%. 16S rRNA gene sequence similarities between the isolate and closely related members of the genus Sphingomonas sensu stricto ranged from 92.6 to 97.3 %, the highest sequence similarities being to Sphingomonas melonis DSM 14444(T) (97.3 %) and Sphingomonas aquatilis DSM 15581(T) (97.3 %). Based on its phenotypic characteristics and the results of DNA-DNA hybridization studies and 16S rRNA gene sequence comparisons, strain BH3(T) represents a novel species of the genus Sphingomonas sensu stricto, for which the name Sphingomonas rubra sp. nov. is proposed. The type strain is BH3(T) ( = CGMCC 1.9113(T) = JCM 16230(T)).

Sphingomonas kaistensis sp. nov., a novel alphaproteobacterium containing pufLM genes

Three Gram-negative, non-motile, non-spore-forming short rods (strains PB56(T), PB180, PB229) were isolated from soil in South Korea. Cells were orange-red in colour. Strains PB180 and PB229 contained small amounts of bacteriochlorophyll a, which was not detected in strain PB56(T). However, all three isolates contained the genes for the photosynthetic type II reaction centre, pufLM. They contained Q-10 as the dominant quinone and C(18 : 1) as the dominant fatty acid. The highest 16S rRNA gene sequence similarities were found to Sphingomonas oligophenolica JCM 12082(T) (95.8 %), Sphingomonas koreensis KCTC 2882(T) (95.1 %), Sphingomonas mali IFO 15500(T) (95.1 %), Sphingomonas faeni DSM 14747(T) (94.8 %), Sphingomonas pruni IFO 15498(T) (94.7 %) and Sphingomonas aquatilis KCTC 2881(T) (94.6 %), as well as to Sphingosinicella microcystinivorans Y2(T) and Sphingosinicella xenopeptidilytica 3-2W4(T) (95.0-95.2 %). Phylogenetic analyses supported the assignment of strains PB56(T), PB180, PB229 to the genus Sphingomonas. The novel isolates differ from all established species of the genus Sphingomonas by their higher G+C content and the absence of straight-chain 2-hydroxy fatty acids. Based on the phylogenetic distances from species with validly published names and their phenotypic properties, the strains constitute a separate species, for which the name Sphingomonas kaistensis sp. nov. is proposed. The type strain is PB56(T) (=KCTC 12334(T)=DSM 16846(T)).

Sphingomonas jaspsi sp. nov., a novel carotenoid-producing bacterium isolated from Misasa, Tottori, Japan

A yellow-pigmented, Gram-negative, motile, strictly aerobic, pleomorphic bacterium (strain TDMA-16(T)) was isolated from a freshwater sample collected at Misasa (Tottori, Japan). Strain TDMA-16(T) was slightly tolerant to gamma-ray irradiation and produced carotenoids, including zeaxanthin, nostoxanthin and an unknown carotenoid, effectively [1.7 mg (g dry cells)(-1)]. The DNA G+C content of strain TDMA-16(T) was 63.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequences placed strain TDMA-16(T) in a distinct lineage in the family Sphingomonadaceae; sequence data showed that strain TDMA-16(T) was most closely related to Sphingomonas mali IFO 15500(T) (95.1 %), Sphingomonas aquatilis JSS7(T) (95.0 %), Sphingomonas pruni IFO 15498(T) (94.9 %), Sphingomonas melonis DSM 14444(T) (94.9 %) and Sphingomonas asaccharolytica IFO 15499(T) (94.5 %). The major fatty acids of strain TDMA-16(T) were C(17 : 1) omega 6c (34.5 %) and C(18 : 1) omega 7c (29.3 %). The presence of Q-10 as the main ubiquinone, the Sphingomonadaceae-specific sphingoglycolipid in the polar lipid profile and 2-hydroxy fatty acids, plus the absence of 3-hydroxy fatty acids, supported identification of this strain as a member of the genus Sphingomonas sensu stricto. Phylogenetic distinctiveness and unique phenotypic characteristics differentiated strain TDMA-16(T) from closely related Sphingomonas species. The results of polyphasic taxonomic analyses suggest that strain TDMA-16(T) represents a novel Sphingomonas species, for which the name Sphingomonas jaspsi sp. nov. is proposed. The type strain is strain TDMA-16(T) (=NBRC 102120(T)=DSM 18422(T)=CCUG 53607(T)).

Description of two novel species, Sphingomonas abaci sp. nov. and Sphingomonas panni sp. nov.

Two Gram-negative, rod-shaped, non-spore-forming bacterial strains designated C42T and C52T were isolated in the Medical Clinic for Small Animals and Ungulates at the University for Veterinary Medicine Vienna, Austria. On the basis of 16S rRNA gene sequence similarity, both strains were shown to belong to the genus Sphingomonas. Strain C42T showed the greatest levels of sequence similarity with Sphingomonas melonis DSM 14444T and Sphingomonas aquatilis KCTC 2881T (both 97.7 %). Strain C52T showed the greatest levels of sequence similarity with Sphingomonas koreensis KCTC 2882T (97.2 %), Sphingomonas aquatilis KCTC 2881T (97.1 %) and S. melonis DSM 14444T (97.0 %). The presence of Q-10 as the main ubiquinone, the predominance of the compound sym-homospermidine in the polyamine patterns, the presence of a Sphingomonadaceae-specific sphingoglycolipid in the polar lipid patterns, the presence of the fatty acid 2-OH C(14 : 0) and the lack of 3-hydroxy fatty acids supported the identification of the two novel strains as members of the genus Sphingomonas sensu stricto. Unique physiological characteristics, protein patterns, quantitative differences in their fatty acid profiles and the results of genomic fingerprinting and DNA-DNA hybridizations differentiated strains C42T and C52T from closely related Sphingomonas species. Hence, the two strains are described as novel species of the genus Sphingomonas sensu stricto. The names Sphingomonas abaci sp. nov. (type strain C42T=LMG 21978T=DSM 15867T) and Sphingomonas panni sp. nov. (type strain C52T=LMG 21979(T)=DSM 15761T) are proposed.