Microbiology

Flavobacterium edaphi sp. nov., isolated from soil from Jeju Island, Korea Abstract An aerobic, Gram-stain-negative, bright yellow-pigmented, oxidase and catalase-positive, non-motile, non-spore forming, rod-shaped strain designated DMN11T was isolated from the soil of crossroads of Jeju Island in South Korea. Colonies were circular, bright yellow-pigmented and smooth with regular edges and measured approximately 1–2 mm in diameter. Flexirubin-type pigments were absent. Phylogenetic tree analysis based on the 16SrRNA gene sequence revealed that the strain DMN11T formed a lineage within the family Flavobacteriaceae of the phylum Bacteroidetes, and it was the most closely related to Flavobacterium suzhouense XIN-1Tand Flavobacterium hauense BX12T (98.6% and 98.2% similarity, respectively). The major isoprenoid quinone was MK-6. The major fatty acids were summed feature 3 (comprising C16:1ω7c and/or C16:1ω6c), iso-C15:0and iso-C15:0 3OH. The polar lipid profile of the strain DMN11T showed the presence of phosphatidylethanolamine (PE) as major lipid. The DNA G+C content was 35.3 mol%, as determined by the thermal denaturation method. The mean levels of DNA–DNA relatedness of the strain DMN11T with F. suzhouense XIN-1T and F. hauense BX12T were 20.5% and 29.2%, respectively. Thus, the data accumulated in this study support the suggestion that the strain DMN11T is considered to represent a novel species of the genus Flavobacterieum, for which the name Flavobacterium edaphi sp. nov. is proposed. The type strain is DMN11T (= KCTC 62114T = JCM 32372T).

The rhizomicrobiomes of wild and cultivated crops react differently to fungicides Abstract The fungicides used to control diseases in cereal production can have adverse effects on non-target microbial communities, with possible consequences for plant health and productivity. Although we know that fungicides affect microbial community structure and soil activities, it is unclear how crop cultivars have altered the impact of fungicides on rhizomicrobiomes. In this study, the rhizosphere bacterial and fungal communities and structures of cultivated crops and their wild relatives were studied by Illumina MiSeq sequencing analysis. The results indicated that the rhizomicrobiome communities of wild crops reacted more strongly to fungicides than that of their cultivated relatives. Furthermore, fungal community composition was more affected by fungicides than bacterial community composition. Remarkably, the same trend was observed in both soybean and rice with regard to the influence of crop cultivar on the response of the rhizomicrobiome to fungicide application, although the level of the response was not similar. We report for the first time that the rhizomicrobiomes of wild crops reacted more strongly to fungicides than the rhizomicrobiomes of cultivated crops.

Caballeronia ginsengisoli sp. nov., isolated from ginseng cultivating soil Abstract A Gram-stain-negative, strictly aerobic, non-motile, ivory colored and rod-shaped bacterium (designated Gsoil 652T) isolated from ginseng cultivating soil, was characterized using a polyphasic approach to clarify its taxonomic position. Strain Gsoil 652T was observed to grow optimally at 30 °C and at pH 7.0 on R2A agar medium. Phylogenetic analysis, based on 16S rRNA gene sequences similarities, indicated that Gsoil 652T belongs to the genus Caballeronia of the family Burkholderiaceae and was most closely related to Caballeronia choica LMG 22940T (98.9%), Caballeronia udeis LMG 27134T (98.9%), Caballeronia sordidicola LMG 22029T (98.2%) and Caballeronia humi LMG 22934T (98.1%). The DNA G+C content was 62.1 mol% and Q-8 was the major isoprenoid quinone. The main polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, unidentified aminophospholipid, and unidentified phospholipid. The predominant fatty acids were C16:0, C17:0 cyclo and C19:0 cyclo ω8c. The DNA–DNA relatedness value between strain Gsoil 652Tand closely related type strains of Caballeronia species were less than 36.0%. Moreover, strain Gsoil 652T could be distinguished phenotypically from the recognized species of the genus Caballeronia. The novel isolate, therefore, represents a novel species, for which the name Caballeronia ginsengisoli sp. nov. is proposed, with the type strain Gsoil 652T (= KACC 19441T = LMG 30326T).

Cinnamaldehyde inhibit Escherichia coli associated with membrane disruption and oxidative damage Abstract In this study, the antimicrobial mechanism of cinnamaldehyde (CIN) against Gram-negative Escherichia coli ATCC 25922 (E. coli) based on membrane and gene regulation was investigated. Treatment with low concentration (0, 1/8, 1/4, 3/8 MIC) of CIN can effectively suppress the growth of E. coli by prolonging its lag phase and Raman spectroscopy showed obvious distinction of the E. coli after being treated with these concentration of CIN. The determination of relative conductivity indicated that CIN at relatively high concentration (0, 1, 2, 4 MIC) can increase the cell membrane permeability, causing the leakage of cellular content. Besides, the content of malondialdehyde (MDA) and the activity of total superoxide dismutase (SOD) of E. coli increased with increasing treatment concentration of CIN, implying that CIN can cause oxidative damage on E. coli cell membrane and induce the increase of total SOD activity to resist this oxidative harm. Moreover, quantitative real-time RT-PCR (qRT-PCR) analysis revealed the relationship between expression of antioxidant genes (SODa, SODb, SODc) and treatment CIN concentration, suggesting that SOD, especially SODc, played a significant role in resistance of E. coli to CIN. The underlying inactivation processing of CIN on E. coli was explored to support CIN as a potential and natural antimicrobial agent in food industry.

Endozoicomonas coralli sp. nov., isolated from the coral Acropora sp. Abstract A novel bacterium, designated strain Acr-12T, was isolated from the coral Acropora sp. off coast of Southern Taiwan. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain Acr-12T belonged to the genus Endozoicomonas and had closest phylogenetic identity to Endozoicomonas acroporae Acr-14T (98.7%) and Endozoicomonas atrinae WP70T (97.8%). Cells of strain Acr-12T were Gram-negative, aerobic, non-motile, poly-β-hydroxybutyrate-accumulating, rod-shaped and formed creamy white colonies. Optimal growth occurred at 30 °C, pH 7, and in the presence of 3% NaCl. Strain Acr-12T contained summed feature 3 (C16:1ω7c and/or C16:1ω6c), summed feature 8 (C18:1ω7c and/or C18:1ω6c) and C16:0 as the predominant fatty acids. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The predominant isoprenoid quinone was Q-9. The genomic DNA G + C content was 49.6 mol%. The DNA–DNA relatedness of strain Acr-12T with respect to the closest species of the genus Endozoicomonas was less than 30%. Phenotypic characteristics of the novel strain also differed from those of the closest related species of the genus Endozoicomonas. On the basis of the genotypic, chemotaxonomic, and phenotypic data, strain Acr-12Trepresents a novel species in the genus Endozoicomonas, for which the name Endozoicomonas coralli sp. nov. is proposed. The type strain is Acr-12T (= BCRC 80921T = KCTC 42900T).

Isolation and identification of Ammodendron bifolium endophytic bacteria and the action mechanism of selected isolates-induced seed germination and their effects on host osmotic-stress tolerance Abstract This study aimed to identify Ammodendron bifolium endophytic bacteria, and to evaluate promoting mechanism of selected isolates on seed germination and their effects on host osmotic-stress tolerance. Forty-five strains were isolated from A. bifolium and were classified into 13 different genera by 16S rDNA gene sequence analysis. AY3, AY9 and AG18, which were identified as Staphylococcus, Kocuria, Bacillus sp., promoted host seed ethylene release during germination. Ethrel and 1-aminocyclopropane-1-carboxylic acid (ACC) imitated the effect of AY3, AY9 and AG18 on seed germination. The data suggest that ethylene mediates AY3-, AY9-, AG18-induced A. bifolium seed germination. In addition, osmotic stress prevented seed germination and radicle elongation. However, the inhibitory effect of osmotic stress on seed germination and radicle elongation were rescued by AY3, AY9 and AG18. The results show that AY3, AY9 and AG18 increased osmotic-stress tolerance in A. bifolium. AY3, AY9, AG18 induced A. bifolium seed germination through promoting ethylene production during endophytic bacteria–plant interaction, and increase osmotic-stress tolerance in A. bifolium. AY3, AY9 and AG18 are potential candidates for the protection of A. bifolium.

Free radical stress induces DNA damage response in RAW264.7 macrophages during Mycobacterium smegmatis infection Abstract Genomic instability resulting from oxidative stress responses may be traced to chromosomal aberration. Oxidative stress suggests an imbalance between the systemic manifestation of reactive free radicals and biological system's ability to repair resulting DNA damage and chromosomal aberration. Bacterial infection associated insult is considered as one of the major factors leading to such stress conditions. To study free radical responses by host cells, RAW 264.7 macrophages were infected with non-pathogenic M. smegmatis mc2155 at different time points. The infection process was followed up with an assessment of free radical stress, cytokine, toll-like receptors (TLRs) and the resulting DNA damage profiles. Results of CFU count showed that maximum infection in macrophages was achieved after 9 h of infection. Host responses to the infection across different time periods were validated from nitric oxide quantification and expression of iNOS and were plotted at regular intervals. IL-10 and TNF-α expression profile at protein and mRNA level showed a heightened pro-inflammatory response by host macrophages to combat M. smegmatis infection. The expression of TLR4, a receptor for recognition of mycobacteria, in infected macrophages reached the highest level at 9 h of infection. Furthermore, comet tail length, micronuclei and γ-H2AX foci recorded the highest level at 9 h of infection, pointing to the fact that breakage in DNA double strands in macrophage reaches its peak at 9 h of infection. In contrast, treatment with ROS inhibitor N-acetyl-l-cysteine (NAC) prevented host cell death through reduction in oxidative stress and DNA damage response during M. smegmatis infection. Therefore, it can be concluded that enhanced oxidative stress response in M. smegmatis infected macrophages might be correlated with DNA damage response.

Modulation of nitrogen metabolism of maize plants inoculated with Azospirillum brasilense and Herbaspirillum seropedicae Abstract Maize is highly responsive to the application of nitrogen to achieve high productivity. Inoculation with diazotrophic bacteria can improve plant growth with low N fertilization. The objective was to evaluate the inoculation of two species of diazotrophs on N metabolism in maize plants, in the presence of two concentrations of nitrogen in a hydroponic system. A factorial arrangement composed of two N levels (3.0 and 0.3 mM), with the presence of Hs—Herbaspirillum seropedicae, and Ab—Azospirillum brasilense or not. The parameters used were dry mass; N, P, and K accumulation; nitrate reductase activity; soluble fractions in roots and leaves. The inoculation altered the N metabolism and promoted greater development of maize plants, as well as a higher accumulation of P and K in the shoots. A more intensive process of N assimilation was evidenced when the plants were inoculated with H. seropedicae, leading to increased levels of NO3− and reduced N-amino, sugars, and NH4+ in leaves associated with high N level, opposite of A. brasilense.

Chromosomal integration of heterologous oxalate decarboxylase in Lactobacillus plantarum WCFS1 using mobile genetic element Ll.LtrB Abstract Lactobacillus plantarum WCFS1 (L. plantarum WCFS1) is commonly used as a potential cell factory because of its 'generally recognized as safe' status. The plasmid instability and the presence of antibiotic selection marker complicate the application of genetically modified L. plantarum in human clinical trials. In the present study, we aimed to integrate oxalate decarboxylase (oxdC) gene of Bacillus subtilis origin by targeted chromosomal mutation in L. plantarum using mobile genetic element Ll.LtrB as a therapeutic tool against calcium oxalate stone disease. oxdC expression cassette was constructed and integrated into a targeted gene, thymidylate synthase (thyA) in the L. plantarum genome. The dependence on external thymidine for growth and survival was established by live dead population assay and SEM (scanning electron microscopy) analysis. The western blotting assay showed the secretion of 44 kDa OxdC protein in the culture supernatant of L. plantarum ∆thyA:OxdC. The biologically contained recombinant strain significantly reduced the oxalate concentration by 53% and exhibited a loss of viability when introduced to environmental samples. Biologically contained L. plantarum secreting OxdC constructed using group II intron has the ability to degrade oxalate present in the extracellular environment and could be used as a therapeutic tool for the calcium oxalate stone disease.

Pharmacological properties of marine macroalgae-associated heterotrophic bacteria Abstract The rich diversity of marine macroalgae and their associated bacterial flora represent a potential reservoir of bioactive compounds with valuable biotechnological and pharmaceutical use. Heterotrophic bacteria associated with the intertidal macroalgae were isolated and evaluated for their pharmacological properties using various in vitro models. Among 148 cultivable isolates, more than 50% were dominated by γ-Proteobacteria and Firmicutes, wherein 53 of them showed consistent antibacterial activity against a broad spectrum of clinically significant pathogens. The bacteria were characterized by extensive microbiological, molecular and chemical identification tools. The heterotrophs Bacillus amyloliquefaciens MTCC 12716 and Shewanella algae MTCC 12715 isolated from a red marine macroalga Hypnea valentiae exhibited potential anti-infective properties against multidrug-resistant pathogens, such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis (minimum inhibitory concentration of 6.25–12.5 µg/mL). The organic extract of B. amyloliquefaciens displayed significantly greater antioxidative properties (IC90 < 1 mg/mL) and the activities showed considerable positive correlation (r2 > 0.8, P < 0.05) with the inhibitory activities against angiotensin converting enzyme-I, pro-inflammatory cyclooxygenases and 5-lipoxygenase, dipeptidyl peptidase-4 and hydroxymethylglutaryl coenzyme A reductase, which were associated with hypertension, inflammation, diabetes, and hypercholesterolemia, respectively. The applications of nuclear magnetic resonance-based fingerprinting to analyze the characteristic signals in the solvent extracts and to correlate them with the pharmaceutical properties were underlined. The heterotrophic bacterium B. amyloliquefaciens MTCC 12716 might, therefore, serve as a potential therapeutic candidate to develop products with wide pharmaceutical applications.