Halalkalibacterium roseum gen. nov., sp. nov., a new member of the family Balneolaceae isolated from soil.

A Gram-stain-negative, non-motile, moderately halophilic and facultatively anaerobic bacterium, designated YR4-1T, was isolated from a saline-alkali and sorghum-planting soil sample collected in Dongying, Shandong Province, PR China. Growth occurred at 28-45 °C with the presence of 4.0-20.0 % (w/v) NaCl and pH 6.0-9.0. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that YR4-1T shared the highest similarity of 92.1-92.4 % with the valid published species of Aliifodinibius. The isolate formed a separate clade at the genus level in recently described family Balneolaceae. The draft genome of strain YR4-1T is 3.83 Mbp long with 44.0 mol% G+C content. The strain possesses several genes involved in the osmotic stress response mechanism and diverse metabolic pathways, probably for the living in saline environment. This may lead to a better understanding of the underrepresented Balneolaceae lineage. The major menaquinone was MK-7. The main polar lipid profile was composed of diphosphatidylglycerol, phosphatidylglycerol, phosphoglycolipids, aminophosphoglycolipid, one glycolipid, and four unidentified lipids. The predominant cellular fatty acids were iso-C15 : 0 (35.7 %) and anteiso-C15 : 0 (33.5 %). On the basis of its phenotypic, chemotaxonomic and phylogenetic features, strain YR4-1T represents a novel species of a new genus, for which the name Halalkalibacterium roseum gen. nov., sp. nov. is proposed. The type strain is YR4-1T (=CGMCC 1.17777T=KCTC 72795T).

Two new Polyangium species, P. aurulentum sp. nov. and P. jinanense sp. nov., isolated from a soil sample.

Polyangium belongs to Polyangiaceae family of Myxococcales, a taxonomic group well-known for their extraordinary social lifestyle and diverse novel gene clusters of secondary metabolites. A yellow-golden strain, designated SDU3-1T, and two rose pink strains, designated SDU13 and SDU14T, were isolated from a soil sample. These three strains were aerobic, mesophilic, not salt-tolerant and were able to prey on living microorganisms. SDU13 and SDU14T formed solitary sporangioles under starvation conditions, while SDU3-1T had no fruiting body structures. They showed 95.9-97.0% (SDU3-1T) or 98.7-98.9% (SDU13 and SDU14T) 16S rRNA gene similarity with the type strains of Polyangium, but were phylogenetically separate from them based on the 16S rRNA gene and genome sequences. Their genomes were 12.3 Mbp (SDU3-1T), 13.9 Mbp (SDU13) and 13.8 Mbp (SDU14T) with the G + C content range of 68.3-69.4 mol%. The average nucleotide identity and DNA-DNA hybridization analyses of genomes further indicated that these three strains belonged to two new species in Polyangium. Their major fatty acids were C18:1ω9c, C16:0 and C18:0. The polyphasic taxonomic characterization suggest that the three strains represent two novel species in the genus Polyangium, for which the names Polyangium aurulentum sp. nov. and Polyangium jinanense sp. nov. are proposed, and the type strains are SDU3-1T (=CGMCC 1.16875T = KCTC 72136T) and SDU14T (=CCTCC AB 2021123T = KCTC 82625T), respectively.

Myxadazoles, Myxobacterium-Derived Isoxazole-Benzimidazole Hybrids with Cardiovascular Activities.

There is a continuous need for novel microbial natural products to fill the drying-up drug development pipeline. Herein, we report myxadazoles from Myxococcus sp. SDU36, a family of novel chimeric small molecules that consist of N-ribityl 5,6-dimethylbenzimidazole and a linear fatty acid chain endowed with an isoxazole ring. The experiments of genome sequencing, gene insertion mutation, isotope labelling, and precursor feeding demonstrated that the fatty acid chain was encoded by a non-canonical PKS/NRPS gene cluster, whereas the origin of N-ribityl 5,6-dimethylbenzimidazole was related to the vitamin B12 metabolism. The convergence of these two distinct biosynthetic pathways through a C-N coupling led to the unique chemical framework of myxadazoles, which is an unprecedented hybridization mode in the paradigm of natural products. Myxadazoles exhibited potent vasculogenesis promotion effect and moderate antithrombotic activity, underscoring their potential usage for the treatment of cardiovascular diseases.

Global Geographic Diversity and Distribution of the Myxobacteria.

Bacteria are globally distributed in various environments on earth, but a global view of the geographic diversity and distribution of a single taxon is lacking. The Earth Microbiome Project (EMP) has established a global collection of microbial communities, providing the possibility for such a survey. Myxococcales is a bacterial order with a potent ability to produce diverse natural products and have wide application potential in agriculture, biomedicine, and environmental protection. In this study, through a comparative analysis of the EMP data and public information, we determined that myxobacteria account for 2.34% of the total bacterial operational taxonomic units (OTUs), and are one of the most diverse bacterial groups on Earth. Myxococcales OTUs are globally distributed and prefer nonsaline soil and sediments, followed by saline environments, but rarely appear in host-associated environments. Myxobacteria are among the least-investigated bacterial groups. The presently cultured and genome-sequenced myxobacteria are most likely environmentally widespread and abundant taxa, and account for approximately 10% and 7% of the myxobacterial community (>97% similarity), respectively. This global panoramic view of the geographic distribution and diversity of myxobacteria, as well as their cultured and genome-sequenced information, will enable us to explore these important bioresources more reasonably and efficiently. The diversity and distribution of myxobacteria beyond the EMP data are further discussed. IMPORTANCE The diversity and distribution of bacteria are crucial for our understanding of their ecological importance and application potential. Myxobacteria are fascinating prokaryotes with multicellular behaviors and a potent capacity for producing secondary metabolites, and have a wide range of potential applications. The ecological importance of myxobacteria in major ecosystems is becoming established, but the global geographic diversity and distribution remain unclear. From a global survey we revealed that Myxococcales OTUs are globally distributed and prefer nonsaline soil and sediments, followed by saline environments, but rarely appear in host-associated environments. The global panoramic view of the geographic distribution and diversity of myxobacteria, as well as their cultured and genome-sequenced information, will enable us to explore these important bioresources more reasonably and efficiently.

The Regulation of LexA on UV-Induced SOS Response in Myxococcus xanthus Based on Transcriptome Analysis.

SOS response is a conserved response to DNA damage in prokaryotes and is negatively regulated by LexA protein, which recognizes specifically an "SOS-box" motif present in the promoter region of SOS genes. Myxococcus xanthus DK1622 possesses a lexA gene, and while the deletion of lexA had no significant effect on either bacterial morphology, UV-C resistance, or sporulation, it did delay growth. UV-C radiation resulted in 651 upregulated genes in M. xanthus, including the typical SOS genes lexA, recA, uvrA, recN and so on, mostly enriched in the pathways of DNA replication and repair, secondary metabolism, and signal transduction. The UV-irradiated lexA mutant also showed the induced expression of SOS genes and these SOS genes enriched into a similar pathway profile to that of wild-type strain. Without irradiation treatment, the absence of LexA enhanced the expression of 122 genes that were not enriched in any pathway. Further analysis of the promoter sequence revealed that in the 122 genes, only the promoters of recA2, lexA and an operon composed of three genes (pafB, pafC and cyaA) had SOS box sequence to which the LexA protein is bound directly. These results update our current understanding of SOS response in M. xanthus and show that UV induces more genes involved in secondary metabolism and signal transduction in addition to DNA replication and repair; and while the canonical LexA-dependent regulation on SOS response has shrunk, only 5 SOS genes are directly repressed by LexA.

Assembly processes and source tracking of planktonic and benthic bacterial communities in the Yellow River estuary.

Estuaries connect rivers with the ocean and are considered transition regions due to the continuous inputs from rivers. Microbiota from different sources converge and undergo succession in these transition regions, but their assembly mechanisms along environmental gradients remain unclear. Here, we found that salinity had a stronger effect on planktonic than on benthic microbial communities, and the dominant planktonic bacteria changed more distinctly than the dominant benthic bacteria with changes in salinity. The planktonic bacteria in the brackish water came mainly from seawater, which was confirmed in the laboratory, whereas the benthic bacteria were weakly affected by salinity, which appeared to be a mixture of the bacteria from riverine and oceanic sediments. Benthic bacterial community assembly in the sediments was mainly controlled by homogeneous selection and almost unaffected by changes in salinity, the dominant assemblage processes for planktonic bacteria changed dramatically along the salinity gradient, from homogeneous selection in freshwater to drift in seawater. Our results highlight that salinity is the key driver of estuarine microbial succession and that salinity is more important in shaping planktonic than benthic bacterial communities in the Yellow River estuary.

Psychroflexus aurantiacus sp. nov., isolated from soil in the Yellow River Delta wetlands.

A Gram-stain-negative, strictly aerobic, non-motile, orange-coloured bacterium, designated YR1-1T, was isolated from a soil sample collected from the Yellow River Delta wetlands (PR China). Growth was observed at a salinity of 1.0-15.0 % NaCl, 4-45 °C and pH 6.0-9.0. The results of phylogenetic analysis based on the 16S rRNA gene sequences indicated that YR1-1T represented a member of the genus Psychroflexus, with the highest sequence similarity to Psychroflexus sediminis YIM-C238T (97.9 %), followed by Psychroflexus aestuariivivens (97.1 %) and Psychroflexus torquis (96.4 %). The average nucleotide identity and digital DNA-DNA hybridization values between YR1-1T and other closely related type strains of species of the genus Psychroflexus were 68.7-86.3% and 17.8-30.9 %. The genome of the strain was 2 899 374 bp in length with 39.8 % DNA G+C content. The predominant fatty acids (>10 %) were iso-C15 : 0 and anteiso-C15 : 0. The major respiratory quinone was menaquinone-6 (MK-6) and the major polar lipids were phosphatidylethanolamine, phospholipid, diphosphatidylglycerol, two unidentified aminolipids and four unidentified lipids. The combined genotypic and phenotypic data indicate that YR1-1T represents a novel species within the genus Psychroflexus, for which the name Psychroflexus aurantiacus sp. nov., is proposed. The type strain is YR1-1T (=KCTC 72794T=CGMCC 1.17458T).

Paucihalobacter ruber gen. nov., sp. nov., isolated from a haloalkaline lake sediment sample.

A Gram-stain-negative, strictly aerobic, non-motile, rod-shaped bacterium, designated CWB-1T, was isolated from a haloalkaline lake sediment sample collected from the bottom of Chaiwopu Lake, Urumchi, Xinjiang Province, PR China. Strain CWB-1T grew at 4-40 °C (optimum, 30-35 °C), pH 6.5-9.0 (optimum, pH 6.5-7.0) and with 0.5-5.5 % (w/v) NaCl (optimum, 2.5-3.0 %). Phylogenetic analyses based on the 16S rRNA gene sequence and the whole genome sequence both revealed that strain CWB-1T belonged to the family Flavobacteriaceae. The strain had the highest similarity of the 16S rRNA gene sequence to Psychroserpens jangbogonensis PAMC 27130T (92.8 %). The genome of strain CWB-1T was 3 548 011 bp long with 36.3 % DNA G+C content. The predominant fatty acids (>10 %) in the CWB-1T cells were iso-C15 : 0, iso-C17 : 0 3-OH and summed feature 1 (iso-C15 : 1 H/C13 : 0 3-OH). The major respiratory quinone was menaquinone-6 and the major polar lipids were phosphatidylethanolamine, an unidentified aminolipid and two unidentified lipids. Based on the phylogenetic analyses, as well as the phenotypic characteristics, a novel genus and species of the family Flavobacteriaceae, Paucihalobacter ruber gen. nov., sp. nov., is proposed. The type strain is CWB-1T (=KCTC 72450T=CGMCC 1.17149T).

Deinococcus terrestris sp. nov., a gamma ray- and ultraviolet-resistant bacterium isolated from soil.

Strain SDU3-2T was isolated from a soil sample collected in Shandong Province, PR China. Cells of SDU3-2T were spherical, Gram-stain-positive, aerobic and non-motile. Cellular growth of the strain occurred at 25-45 °C, pH 5.5-8.5 and with 0-1.5 % (w/v) of NaCl. Phylogenetic analysis based on the 16S rRNA gene sequences showed that strain SDU3-2T was closest to the type strain Deinococcus murrayi ALT-1bT with a similarity of 95.2 %. The draft genome was 3.49 Mbp long with 69.2 mol% G+C content. Strain SDU3-2T exhibited high resistance to gamma radiation (D10 >12 kGy) and UV (D10 >900 J m-2). The strain encoded many genes for resistance to radiation and oxidative stress, which were highly conserved with other Deinococcus species, but possessed interspecific properties. The major fatty acids of SDU3-2T cells were C15 : 1 ω6c, C16 : 1 ω7c/C16 : 1 ω6c, and C17 : 1 ω8c, the major menaquinone was menaquinone-8, and the major polar lipids were an unidentified phosphoglycolipid, four unidentified glycolipids and an unidentified phospholipid. The average nucleotide identity and DNA-DNA hybridization results further indicated that strain SDU3-2T represents a new species in the genus Deinococcus, for which the name Deinococcus terrestris sp. nov. is proposed. The type strain is SDU3-2T (=CGMCC 1.17147T=KCTC 43098T).

Genes involved in fatty acid metabolism: molecular characterization and hypothalamic mRNA response to energy status and neuropeptide Y treatment in the orange-spotted grouper Epinephelus coioides.

As in mammals, fatty acid (FA) metabolism plays diverse and vital roles in regulating food intake in fish. Multiple lines of evidence suggest that the effect of FA metabolism on food intake is linked to changes in the level of neuropeptide Y (NPY) in the hypothalamus of the rainbow trout. In mammals, the evidence suggests that FA metabolism regulates feeding via hypothalamic NPY. NPY is therefore considered an important factor that mediates the modulation of food intake by FA metabolism in vertebrates. The stimulatory effect of NPY on food intake is well known. However, to the best of our knowledge, the effect of NPY on FA metabolism in the hypothalamus has not been examined. In this study, we cloned the cDNA of four key enzymes involved in FA metabolism and assessed the effect of energy status and NPY on their mRNA expression in the hypothalamus of grouper. The full-length cDNAs of UCP2 and CPT1a and the partial coding sequence (CDS) of ACC1 and FAS were isolated from the grouper hypothalamus. These genes are expressed in the hypothalamus and during the organogenetic stage of embryogenesis. A feeding rhythm study showed that the hypothalamic expression level of NPY and CPT1a was highly correlated with feeding rhythm. Long-term fasting was found to significantly induce the hypothalamic mRNA expression of NPY, CPT1a and UCP2. An in vitro study demonstrated that NPY strongly stimulated CPT1a and UCP2 mRNA expression in a time- and dose-dependent manner. Collectively, these results suggest that these four genes related to FA metabolism may play a role in regulating food intake in grouper and, that NPY modulates FA metabolism in the grouper hypothalamus. This study showed, for the first time in vertebrates, the effect of NPY on the gene expression of FA metabolism-related enzymes.

Stimulatory effects of neuropeptide Y on the growth of orange-spotted grouper (Epinephelus coioides).

Neuropeptide Y (NPY) is a member of the pancreatic polypeptide family which is a potent orexigenic peptide known to date in mammals and teleost. This study was carried out to investigate the effects of NPY on food intake and growth of orange-spotted grouper (Epinephelus coioides). Synthetic grouper NPY (gNPY) was given orally at the dose of 0.5, 1.0 and 2.0 µg/g feed for 50 days, results showed that NPY treatment (1.0 and 2.0 µg/g feed) significantly increased growth rate, weight gain, feed conversion efficiency (FCE) and pituitary growth hormone (GH) mRNA level than the control group (p<0.05). Furthermore, high level secretion of gNPY was expressed and purified in the Pichia pastoris expression system. The bioactivity of recombinant gNPY was confirmed by its ability to up-regulate GH mRNA expression in vivo and in vitro and down-regulate preprosomatostatin I (PSSI) mRNA expression in vivo. These results demonstrate that NPY has stimulatory effects on food intake as well as growth of grouper as in other teleost fish, also indicate that recombinant gNPY from P. pastoris has the same bioactivity as synthetic gNPY and has the potential to be used as a feed additive for both research and aquatic application.

Expression of insulin-like growth factor-1 of orange-spotted grouper (Epinephelus coioides) in yeast Pichia pastoris.

IGF-1 plays a key role in development, growth, and metabolism in teleost. Recombinant fish IGF-1 may be a useful tool for both theoretical research and aquaculture applications. However, using the Escherichia coli expression system has several drawbacks for producing quality fish IGF-1 protein. To explore the yeast expression system for generating fish IGF-1 protein, the cDNA coding for the mature orange-spotted grouper IGF-1 peptide without signal peptide and E domain was cloned into the secreting expression organism Pichia pastoris. Tricine-SDS-PAGE and western blotting analysis of culture medium from methanol-induced expression yeast clones demonstrated that the rgIGF-1 was secreted into the culture medium, had a molecular weight of 8.7 kDa. The production peaked at 24h of induction and the optimal pH for expression was 5.0. The recombinant protein was purified using a combined ammonium sulfate precipitation with Ni(2+) affinity chromatography. Finally, 17.9 mg of the protein was obtained from 420 ml of the culture supernatant and the purity was about 92.4%. Bioactivity of the rgIGF-1 was confirmed by the ability to stimulate proliferation of embryo cell line of grouper (GP cell line) and MFC-7 cell. The present results suggest that the Pichia pastoris expression system can be used to produce a functional rgIGF-1 for both research and aquaculture application.