Phenylobacterium montanum sp. nov., an oligotrophic, slightly acidophilic mesophile isolated from sandy soil.

A bacterial strain, designated S6T, was isolated from the sandy soil on a rocky mountain in South China. Cells of S6T were Gram-stain-negative, aerobic, non-spore-forming, non-motile and non-prosthecae-producing. 16S rRNA gene sequence analysis revealed the highest similarities to 12 uncultured bacteria, followed by Phenylobacterium sp. B6.10-61 (97.14 %). The closest related validly published strains are Caulobacter henricii ATCC 15253T (96.15 %), Phenylobacterium conjunctum FWC 21T (96.08 %) and Caulobacter mirabilis FWC 38T (96.08 %). Phylogenetic analysis based on 16S rRNA gene, genome and proteome sequences demonstrated that S6T formed a separated lineage in the genus Phenylobacterium. Strain S6T contained Q-10 (97.5 %) as the major ubiquinone and C18 : 1 ω7c and C16 : 0 as the major fatty acids. The polar lipid profile consisted of phosphatidylglycerol, an unknown phosphoglycolipid and three unknown glycolipids. The assembled genome comprises a chromosome with a length of 5.5 Mb and a plasmid of 96 014 bp. The G+C content was 67.6 mol%. The morphological, physiological, chemotaxonomic and phylogenetic analyses clearly distinguished this strain from its closest phylogenetic neighbours. Thus it is proposed that strain S6T represents a novel species in the genus Phenylobacterium, for which the name Phenylobacterium montanum sp. nov. is proposed. The type strain is S6T (=NBRC 115419T=GCMCC 1.18594T).

First report of Agroathelia rolfsii causing southern blight on cowpea in China.

Cowpea (Vigna unguiculata L.) is a legume consumed as a high-quality plant protein source in many parts of the world. In August 2023, it was observed that a plant disease affected cowpea growth in Yiyang (28.34°N, 112.55°E), China. The average disease incidence was 10%, resulting in 8.5% economic losses in approximately 3,000 m2. The symptoms initially appeared as brown lesions near the stem-soil interface and the lesions were colonized by white mycelia. As the disease progressed, the disease symptoms included constriction and brown staining at the base of the stem, covered with a small amount of white mycelia. Eventually, the entire plants withered and collapsed and many sclerotia were scattered on the ground around the diseased stem. Twenty samples (10 sclerotia and 10 diseased tissue fragments) were collected from symptomatic plants for causal agent isolation. Samples were disinfected with 70% ethanol for 30 s, 5% NaClO for 1 min, rinsed three times with sterile water, dried and placed on potato dextrose agar (PDA) plates at 28℃ in the dark. In total, 20 isolates were obtained by the hyphal tip method (Terrones et al. 2022) and showed a consistent phenotype of white cottony mycelia on PDA with an growth rate of 12.9 to 21.3 mm/day (n = 20). Sclerotia formed at five to eight days post inoculation, were initially whitish, turning beige and eventually dark brown. The diameter of mature sclerotia ranged from 0.89 to 2.13 mm (mean = 1.64±0.29 mm; n =50). For pathogen identification, ITS1/ITS4 (White et al. 1990) and EF1-983F/EF1-2218R (Rehner and Buckley 2005) primers were used to amplify the internal transcribed spacer regions (ITS) and translation elongation factor-1 alpha gene (TEF-1α), respectively. The sequences of all 20 isolates showed 99% to 100% similarity with Agroathelia rolfsii sequences from GenBank by BLAST analysis. The sequences of two representative strains, ID1 and ID4, were deposited in GenBank. The ITS sequences of ID1 (OR689482) and ID4 (OR689481) were ＞99% similar to A. rolfsii strain QJ7 (593/596 bp; MZ750983) and A. rolfsii strain Kale078 (565/568 bp; MN872304), respectively. Also, TEF-1α sequences of ID1 (OR713735) and ID4 (OR713736) were ＞99% similar to the sequences of A. rolfsii strain HS-Sr (1073/1073 bp; OL416131) and A. rolfsii strain MSB1-2 (1070/1075 bp; MN702790), respectively. Phylogenetic analysis based on ITS and TEF1-α sequences indicated that ID1 and ID4 clustered into the A. rolfsii clade. Based on morphology and sequence analyses, the isolates ID1 and ID4 were identified as A. rolfsii (anamorph Sclerotium rolfsii). Pathogenicity tests were conducted three times on healthy 30-day-old cowpea seedlings. Five plants were inoculated with 6-day-old mycelial discs (6 mm) of ID1 or ID4 at the base of the seedlings (n = 30) while four plants were inoculated with a sterile PDA disc as a control (n = 12). All seedlings were cultivated in a greenhouse with a temperature of 26°C to 28°C and relative humidity 60% to 80% with a 14/10 h light/dark photoperiod. Eight days later, all the fungal inoculated seedlings showed symptoms including brown necrosis and collapse of the stems, and eventual withering of the seedlings. Control plants remained asymptomatic. The causal pathogens were reisolated in PDA plates and identified by ITS sequence analysis, completing Koch's postulates. To our knowledge, this is the first report of A. rolfsii causing southern blight on cowpea in China. Early accurate diagnosis will help farmers to adopt suitable practices to control disease outbreaks and reduce losses.

Polyamine-containing natural products: structure, bioactivity, and biosynthesis.

Covering: 2005 to August, 2023Polyamine-containing natural products (NPs) have been isolated from a wide range of terrestrial and marine organisms and most of them exhibit remarkable and diverse activities, including antimicrobial, antiprotozoal, antiangiogenic, antitumor, antiviral, iron-chelating, anti-depressive, anti-inflammatory, insecticidal, antiobesity, and antioxidant properties. Their extraordinary activities and potential applications in human health and agriculture attract increasing numbers of studies on polyamine-containing NPs. In this review, we summarized the source, structure, classification, bioactivities and biosynthesis of polyamine-containing NPs, focusing on the biosynthetic mechanism of polyamine itself and representative polyamine alkaloids, polyamine-containing siderophores with catechol/hydroxamate/hydroxycarboxylate groups, nonribosomal peptide-(polyketide)-polyamine (NRP-(PK)-PA), and NRP-PK-long chain poly-fatty amine (lcPFAN) hybrid molecules.

First Report of Southern Blight Caused by Athelia rolfsii on Pepper in Yiyang, China.

Pepper(Capsicum annuum L.) is the vegetable with the largest production area China (Zou and Zou 2021). In the summer of 2020 and 2021, disease symptoms were observed in C. annuum L. cv. bola in a 10-ha field in Yiyang(28.35°N, 112.56°E), Hunan province of China. The disease incidence ranged from 10% to 30%. The symptoms initially appeared as tan lesions, which were colonized by fast-growing white mycelia, at the soil line. Affected plants eventually became wilted. Wilting was accompanied by girdling of the stem at the base, and signs of the pathogen, mycelia and golden-brown colored sclerotia. The spatial distribution of the disease was either single plants or small foci of affected plants. Diseased stem sections (1.0~1.5 cm) of 20 plants from the field in 2021 with typical symptoms were surface sterilized with 75% ethanol for 30 s, followed by 60 s in 2.5% NaClO, rinsed thrice with sterile water, air dried and plated on potato dextrose agar (PDA), and incubated at 28℃ in the dark for 5 days to isolate the causative pathogen. Twenty fungal isolates with similar colony morphology were collected and purified. These isolates formed radial colonies, and abundant sclerotia were observed after 5 to 10 days of incubation at 28℃. The color of the sclerotia with a diameter of 1.39 ± 0.15 mm (1.15 to 1.60, n=50) gradually changed from white to light yellow, and finally to dark brown. The representative isolate YYBJ20 was selected for further molecular identification. The internal transcribed spacer region and elongation factor-1alpha gene were amplified using the primers, ITS1/ITS4 (White et al. 1990) and EF1-983F/EF1-2218R (Rehner and Buckley 2005), respectively. The ITS and EF1α amplicons were sequenced and deposited in GenBank with the accession numbers OQ186649 and OQ221158, respectively. Sequence analysis revealed that the ITS and EF1α sequences of the YYBJ20 isolate exhibited ≥99% of identity with the ITS (MH260413 and AB075300) and EF1α (OL416131 and MW322687) sequences of Athelia rolfsii, respectively. Phylogenetic analysis classified YYBJ20 into a common clade with different A. rolfsii strains, but different from other Athelia or Sclerotium species. For pathogenicity tests, PDA plugs (6 mm diam.) colonized by 3-day-old mycelia were inoculated into the stem bases of 30-day-old pepper seedlings (n=10). Another 10 seedlings were inoculated with noncolonized PDA plugs were used as noninoculated controls. The pepper seedlings were incubated at 28 ± 2℃ and 60 to 80 % relative humidity under a 14h-10h of light-dark cycle. After 10 days of incubation, ten YYBJ20-inoculated plants were wilted with similar symptoms to those observed in the field, while control plants remained healthy. The pathogenicity tests were repeated three times. The fungal strain re-isolated from the infected seedlings (100% re-isolation frequency) showed the same morphological and molecular traits as the original isolates from the diseased plants. No fungi were isolated from the control plants, which is consistent with the Koch's postulates. Based on the morphological and sequencing results, the causative fungus was identified as A. rolfsii (anamorph Sclerotium rolfsii). To our knowledge, this is the first report of A. rolfsii causing southern blight on pepper in China. Due to the broad host range of and serious consequences caused by A. rolfsii (Lei et al. 2021; Zhang et al. 2022; Zhu et al. 2022), this research will be beneficial to develop strategies to mitigate future losses of pepper in China.

Characterization of a pathway-specific activator of edeine biosynthesis and improved edeine production by its overexpression in Brevibacillus brevis.

Edeines are a group of non-ribosomal antibacterial peptides produced by Brevibacillus brevis. Due to the significant antibacterial properties of edeines, increasing edeine yield is of great interest in biomedical research. Herein, we identified that EdeB, a member of the ParB protein family, significantly improved edeine production in B. brevis. First, overexpression of edeB in B. brevis X23 increased edeine production by 92.27%. Second, in vitro bacteriostasis experiment showed that edeB-deletion mutant exhibited less antibacterial activity. Third, RT-qPCR assay demonstrated that the expression of edeA, edeQ, and edeK, which are key components of the edeine biosynthesis pathway, in edeB-deletion mutant X23(ΔedeB) was significantly lower than that in wild-type B. brevis strain X23. Finally, electrophoretic mobility shift assay (EMSA) showed that EdeB directly bound to the promoter region of the edeine biosynthetic gene cluster (ede BGC), suggesting that EdeB improves edeine production through interaction with ede BGC in B. brevis.

Enhancement of edeine production in Brevibacillus brevis X23 via in situ promoter engineering.

Edeines, a group of cationic antimicrobial peptides produced by the soil bacterium Brevibacillus, have broad biological effects, such as antimicrobial, anticancer and immunosuppressive activities. However, the yield of edeines in wild-type (WT) Brevibacillus is extremely low, and chemical synthesis of edeines is a time-consuming process. Genetic engineering has proven to be an effective approach to produce antibiotics with high yield. In this study, the edeine biosynthetic gene cluster (ede BGC), which is involved in edeine production, was identified and characterized in Brevibacillus brevis X23. To improve edeine production in B. brevis X23, the ede BGC promoter was replaced with six different promoters, Pmwp , Pspc , PxylA , Pshuttle-09 , Pgrac or P43 , through double-crossover homologous recombination. The new promoters significantly increased the expression of the ede BGC as well as edeine production by 2.9 ± 0.4 to 20.5 ± 1.2-fold and 3.6 ± 0.1to 8.7 ± 0.7-fold respectively. The highest yield of edeines (83.6 mg l-1 ) was obtained in B. brevis X23 with the Pmwp promoter. This study provides a practical approach for producing high yields of edeines in B. brevis.

The Discovery of Actinospene, a New Polyene Macrolide with Broad Activity against Plant Fungal Pathogens and Pathogenic Yeasts.

Phytopathogenic fungi infect crops, presenting a worldwide threat to agriculture. Polyene macrolides are one of the most effective antifungal agents applied in human therapy and crop protection. In this study, we found a cryptic polyene biosynthetic gene cluster in Actinokineospora spheciospongiae by genome mining. Then, this gene cluster was activated via varying fermentation conditions, leading to the discovery of new polyene actinospene (1), which was subsequently isolated and its structure determined through spectroscopic techniques including UV, HR-MS, and NMR. The absolute configuration was confirmed by comparing the calculated and experimental electronic circular dichroism (ECD) spectra. Unlike known polyene macrolides, actinospene (1) demonstrated more versatile post-assembling decorations including two epoxide groups and an unusual isobutenyl side chain. In bioassays, actinospene (1) showed a broad spectrum of antifungal activity against several plant fungal pathogens as well as pathogenic yeasts with minimum inhibitory concentrations ranging between 2 and 10 µg/mL.

Complete Genome Sequence of Caulobacter sp. Strain S6, Obtained Using Oxford Nanopore and Illumina Sequencing.

We report the complete genome sequence of Caulobacter sp. strain S6, generated from Oxford Nanopore and Illumina sequencing. The assembled genome comprises a chromosome with a length of 5.5 Mb and a plasmid of 96,014 bp.

Complete Genome Sequence of Sandaracinobacter sp. Strain M6, Isolated from a Rocky Mountain in China.

We report the complete genome sequence of Sandaracinobacter sp. strain M6 obtained by Oxford Nanopore and Illumina sequencing. The approximately 3.4-Mb genome sequence with a GC content of 67.65% provides essential data for future taxonomic studies and information for further investigating the metabolic characteristics of aerobic anoxygenic phototrophs.

Optimization, Characteristics, and Functions of Alkaline Phosphatase From Escherichia coli.

Weaning of piglets could increase the risk of infecting with Gram-negative pathogens, which can further bring about a wide array of virulence factors including the endotoxin lipopolysaccharide (LPS). It is in common practice that the use of antibiotics has been restricted in animal husbandry. Alkaline phosphatase (AKP) plays an important role in the detoxification and anti-inflammatory effects of LPS. This study investigated the protective effects of AKP on intestinal epithelial cells during inflammation. Site-directed mutagenesis was performed to modulate the AKP activity. The enzyme activity tests showed that the activity of the DelSigD153G-D330N mutants in B. subtilis was nearly 1,600 times higher than that of the wild-type AKP. In this study, an in vitro LPS-induced inflammation model using IPEC-J2 cells was established. The mRNA expression of interleukin-(IL-) 6, IL-8, and tumor necrosis factor-α (TNF-α) were extremely significantly downregulated, and that of ASC amino acid transporter 2 (ASCT-2), zonula occludens protein-1 (ZO-1), and occludin-3 (CLDN-3) were significantly upregulated by the DelSigD153G-D330N mutant compared with LPS treatment. This concludes the anti-inflammatory role of AKP on epithelial membrane, and we are hopeful that this research could achieve a sustainable development for the pig industry.

An automatic segmentation method for heart sounds.

BACKGROUND: There are two major challenges in automated heart sound analysis: segmentation and classification. An efficient segmentation is capable of providing valuable diagnostic information of patients. In addition, it is crucial for some feature-extraction based classification methods. Therefore, the segmentation of heart sound is of significant value. METHODS: This paper presents an automatic heart sound segmentation method that combines the time-domain analysis, frequency-domain analysis and time-frequency-domain analysis. Employing this method, the boundaries of heart sound components are first located, and the components are then recognized. Finally, the heart sounds are divided into several segments on the basis of the results of boundary localization and component identification. RESULTS: In order to evaluate the performance of the proposed method, quantitative experiments are performed on an authoritative heart sound database. The experimental results show that the boundary localization has a sensitivity (Se) of 100%, a positive predictive value (PPV) of 99.3% and an accuracy (Acc) of 99.93%. Moreover, the Se, PPV and Acc of component identification reach 98.63, 99.86 and 98.49%, respectively. CONCLUSION: The proposed method shows reliable performance on the segmentation of heart sounds. Compared with previous works, this method can be applied to not only normal heart sounds, but also the sounds with S3, S4 and murmurs, thus greatly increasing the applied range.

Environmental factors shaping the diversity of bacterial communities that promote rice production.

BACKGROUND: Exploiting soil microorganisms in the rhizosphere of plants can significantly improve agricultural productivity; however, the mechanism by which microorganisms specifically affect agricultural productivity is poorly understood. To clarify this uncertainly, the rhizospheric microbial communities of super rice plants at various growth stages were analysed using 16S rRNA high-throughput gene sequencing; microbial communities were then related to soil properties and rice productivity. RESULTS: The rhizospheric bacterial communities were characterized by the phyla Proteobacteria, Acidobacteria, Chloroflexi, and Verrucomicrobia during all stages of rice growth. Rice production differed by approximately 30% between high- and low-yield sites that had uniform fertilization regimes and climatic conditions, suggesting the key role of microbial communities. Mantel tests showed a strong correlation between soil conditions and rhizospheric bacterial communities, and microorganisms had different effects on crop yield. Among the four growing periods, the rhizospheric bacterial communities present during the heading stage showed a more significant correlation (p <  0.05) with crop yield, suggesting their potential in regulating crop production. The biological properties (i.e., microbes) reflected the situation of agricultural land better than the physicochemical characterics (i.e., nutrient elements), which provides theoretical support for agronomic production. Molecular ecological network (MEN) analysis suggested that differences in productivity were caused by the interaction between the soil characteristics and the bacterial communities. CONCLUSIONS: During the heading stage of rice cropping, the rhizospheric microbial community is vital for the resulting rice yield. According to network analysis, the cooperative relationship (i.e., positive interaction) between between microbes may contribute significantly to yield, and the biological properties (i.e., microbes) better reflected the real conditions of agricultural land than did the physicochemical characteristics (i.e., nutrient elements).

Yield improvement of epothilones in Burkholderia strain DSM7029 via transporter engineering.

Transporter engineering has been shown to be a positive approach for enhancing natural product titers in microbial cell factories by expelling target compounds out of feasible hosts. In this work, two multidrug efflux pumps, Orf14 and Orf3, were modulated in the epothilone production strain Burkholderia DSM7029::Tn5-km-epo (named G32) via Red/ET engineering to increase heterologous polyketide epothilone yields. Compared with the prior G32 strain, the total production of several epothilones in the G32::orf14-orf3 mutant was meaningfully doubled according to high-performance liquid chromatography-mass spectrometer analysis. Typically for epothilone B, in simple and clear liquid medium CYMG, the overall productivity in the engineered high-yield producer G32::orf14-orf3 was improved for almost 3-fold, from 2.7 to about 8.1 µg/l. Additionally, the ratio of extracellular to intracellular accumulation of epothilone B was raised from 9.3:1 to 13.7:1 in response to expression of two putative transport genes orf14 and orf3. Hence, we strongly recommend that the Orf14 and Orf3 transporters export epothilone, thus promotes the forward reaction of biosynthesis on epothilone manufacture inside the cells. Our results afford a practical stage for yield improvement of other heterologous natural products in broad chassis cells.

Simple and rapid direct cloning and heterologous expression of natural product biosynthetic gene cluster in Bacillus subtilis via Red/ET recombineering.

Heterologous expression of biosynthetic pathways is an important way to research and discover microbial natural products. Bacillus subtilis is a suitable host for the heterologous production of natural products from bacilli and related Firmicutes. Existing technologies for heterologous expression of large biosynthetic gene clusters in B. subtilis are complicated. Herein, we present a simple and rapid strategy for direct cloning based heterologous expression of biosynthetic pathways in B. subtilis via Red/ET recombineering, using a 5.2 kb specific direct cloning vector carrying homologous sequences to the amyE gene in B. subtilis and CcdB counterselection marker. Using a two-step procedure, two large biosynthetic pathways for edeine (48.3 kb) and bacillomycin (37.2 kb) from Brevibacillus brevis X23 and B. amyloliquefaciens FZB42, respectively, were directly cloned and subsequently integrated into the chromosome of B. subtilis within one week. The gene cluster for bacillomycin was successfully expressed in the heterologous host, although edeine production was not detectable. Compared with similar technologies, this method offers a simpler and more feasible system for the discovery of natural products from bacilli and related genera.

Decolorization and degradation of reactive dye during the dyed cotton fabric rinsing process.

Dyeing process of textile consumes large quantities of water, which results in huge amounts of colored wastewater. Most of the dye wastewater treating methods focused on the treatment of wastewater after the rinsing process of dyed textile. In this paper, tetraacetylethylenediamine/hydrogen peroxide (TAED/H2O2) active oxidation (AO) system was developed to rinse dyed textile and decolorize the rinsing wastewater simultaneously. The results indicated that the decolorization ratio of the rinse effluent obtained by AO method were in the range of 51.72%-84.15% according to different dyes and the COD value decreased more than 30% compared with that of traditional rinsing process. The decolorization kinetics investigation showed that the decolorization of dyes during AO rinsing process followed the law of pseudo-first order kinetics. The result of UV-Vis and UPLC-MS analysis demonstrated that the dye was degraded into colorless organic molecular fragments and partly mineralized during the AO rinsing process.