The Draft Genome Sequence of Methylophilus sp. D22, Capable of Growing Under High Concentration of Methanol Abstract In this study, a wild type Methylophilus sp. strain D22 belonging to the family Methylophilus was isolated and characterized, which shows high tolerance towards methanol, as it can grow under 50 g/L of methanol. Methylophilus sp. strain D22 was isolated from the lake sludge in Nanjing Tech University, China. The assembled draft genome contains one circular chromosome with 3,004,398 bp, 49.7% of GC content, and 2107 predicted encoding proteins. Sequence-based genomic analysis demonstrates that the assimilation pathway of ribulose monophosphate (RuMP) pathway and dissimilation pathway of tetrahydromethanopterin (H4MPT) pathway are co-existing and contribute to the high methanol utilization efficiency.

Spiking a Silty-Sand Reference Soil with Bacterial DNA: Limits and Pitfalls in the Discrimination of Live and Dead Cells When Applying Ethidium Monoazide (EMA) Treatment Abstract In the present study, EMA (ethidium monoazide) treatment was applied to a silty-sand reference soil prior to DNA extraction to enable a differentiation between dead and living cells. For this purpose, a reference soil was spiked with Listeria monocytogenes cells or cell equivalents, respectively. With the purpose of evaluating optimum treatment conditions, different EMA concentrations have been tested. However, the results remained largely inconclusive. Furthermore, varied dark incubation periods allowing EMA to penetrate dead cells did not allow the selective removal of DNA from membrane-compromised cells in downstream analyses. In contrast to undiluted soil, an effect of EMA treatment during DNA extraction could be observed when using a 1:10 dilution of the reference soil; however, the effect has not been sufficiently selective to act on heat-treated cells only. Although the application of EMA to soil requires further evaluation, the procedure harbors future potential for improving DNA-based approaches in microbial ecology studies.

Comparison of Starvation-Induced Persister Cells with Antibiotic-Induced Persister Cells Abstract The phenotypic heterogeneity in a large population arises because of fluctuation in microenvironments and stochastic gene expressions. In this report, we isolated two types of persistent sub-populations of Vibrio cholerae, one triggered by starvation and another by antibiotics. We characterised starvation-induced (E-cells) and antibiotic-induced (P-cell) persister cells for stress tolerance, colony morphology and toxin gene expressions. Both the sub-populations differ with respect to morphology, temperature tolerance and oxidative stress tolerance. The E-cells were smaller than the P-cells and formed tiny colonies (1–2 mm). The E-cells were more sensitive to heat and oxidative stress compared with P-cells. The up-regulated genes of P-cells include, genes of antioxidant enzymes (>5 fold), cholera toxin (>26 fold) and toxin: antitoxin protein hipA (>100 fold). Upon nutrient up-shift, the E-cells recovered after lag time of 6 h. However, such lag extension was not visible during P-cell recovery, suggesting that P-cell physiology is more akin to normal cells than E-cells. This is the first comparative report on the two different persister sub-populations of V. cholerae. The E-cells and P-cells are similar regarding antibiotic tolerance. However, the sub-populations differ significantly in stress tolerance and other phenotypes studied.

Functional Characterization of Novel U6 RNA Polymerase III Promoters: Their Implication for CRISPR-Cas9-Mediated Gene Editing in Aspergillus oryzae Abstract U6 RNA polymerase III promoter (PU6), which is a key element in controlling the generation of single-guide RNA (sgRNA) for gene editing through CRISPR-Cas9 system, was investigated in this work. Using bioinformatics approach, two novel U6 ribonucleic acid (U6 RNA) sequences of Aspergillus niger were identified, showing that they had conserved motifs similar to other U6 RNAs. The putative PU6 located at the upstream sequence of A. niger U6 RNA exhibited the consensus motif, CCAATYA, and the TATA box which shared highly conserved characteristics across Aspergilli, whereas the A- and B-boxes were found at the intragenic and downstream of the structural genes, respectively. Using Aspergillus oryzae as a workhorse system, the function of A. niger PU6s for controlling the transcripts of sgRNA was verified, in which the orotidine-5′-phosphate decarboxylase (pyrG) sequence was used as a target for gene disruption through CRISPR-Cas9 system. Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) analysis of the selected pyrG auxotrophic strains showed the expression of sgRNA, indicating that the non-native promoters could efficiently drive sgRNA expression in A. oryzae. These identified promoters are useful genetic tools for precise engineering of metabolic pathways in the industrially important fungus through the empowered CRISPR-Cas9-associated gene-editing system.

Glycine Betaine Effect on Dormancy in Deinococcus sp. UDEC-P1 and Psychrobacter sp. UDEC-A5 Exposed to Hyperosmotic Stress Abstract Bacteria under stress increase the proportion of dormant cells to ensure their survival. Cold and osmotic stress are similar, because in both the availability of water is reduced. Glycine betaine (GB) is one of the most common osmoprotectants in bacteria and possesses cryoprotectant properties. Our aim was to determine whether GB modifies the proportion of dormant Deinococcus sp. UDEC-P1 and Psychrobacter sp. UDEC-A5 cells exposed to osmotic stress. Both bacterial strains were incubated in the presence of up to 1 M NaCl with or without GB. Active and dormant cells were evaluated by both spectrophotometric and flow cytometry analysis. Without GB, Deinococcus sp. UDEC-P1 grew in the presence of 0.05 M NaCl, but with 5 mM GB grew at 0.1 M NaCl. Psychrobacter sp. UDEC-A5 grew in the presence of up to 0.25 M NaCl, but with 5 mM GB grew at 0.5 M NaCl. Under osmotic stress, the proportion of dormant cells of Deinococcus sp. UDEC-P1 and Psychrobacter sp. UDEC-A5 increased significantly (about eightfold and fivefold, respectively). The addition of GB (5 mM) exerted a different effect on the two strains, since it avoided the entrance into the dormancy of Psychrobacter sp. UDEC-A5 cells, but not of Deinococcus sp. UDEC-P1 cells. Our results suggest that the effect of GB on bacterial metabolism is strain dependent. For bacteria in which GB avoids dormancy, such as Psychrobacter sp. UDEC-A5, it could be a “double-edged sword” by reducing the “seed bank” available to recover the active population when favorable conditions return.

Pontibacter beigongshangensis sp. nov., Isolated from the Mash of Wine Abstract A Gram-negative, aerobic, oval-shaped, and light red pigmented bacterium, designated T6-1T, was isolated from the mash of wine collected from a wine-making laboratory simulated fermenter located in Beijing, China. The optimal growth of T6-1T occurred at 30 °C, pH 7.0 with 1% NaCl. The sole respiratory quinone was menaquinone-7 (MK-7). The principal cellular fatty acids (>5%) were iso-C15:0, iso-C17:0 3OH, C16:1 ω5c, and iso-C17:0. The major polar lipids were PE (phosphatidylethanolamine), PL (unidentified phospholipid), and L1-2 (unidentified lipids). 16S rRNA phylogenetic analysis indicated that strain T6-1T belonged to the genus Pontibacter. The 16S rRNA gene sequence of strain T6-1T was most similar to Pontibacter amylolyticus 9-2T (95.92%). The genomic DNA G+C content of strain T6-1 was 50.34 mol%. The digital DNA–DNA relatedness and average nucleotide identity value between T6-1T and 9-2T was 20.20% and 74.18%, respectively. Polyphasic taxonomy analysis indicated that strain T6-1T represents a novel species of the genus Pontibacter, for which the name Pontibacter beigongshangensis sp. nov. is proposed, with the type strain T6-1T (= CGMCC 1.17104T = KCTC 72413T).

Diversity of Archaea and Its Correlation with Environmental Factors in the Ebinur Lake Wetland Abstract The diversity and community composition of archaea in soil samples from three wetlands (SP1, SP2, and SP3) of Ebinur Lake were studied by constructing 16S rDNA cloning library. The correlation between the diversity of archaea and soil environmental factors was analyzed by CANOCO software. The aim of this study was to reveal the differences of community structures of archaea in different sample sites, to provide a theoretical basis for further study on degradation and restoration of Ebinur Lake wetland. The results showed that Euryarchaeota accounted for 57.1% was the most dominant phylum observed, followed by Thaumarchaeota and Crenarchaeota for the three wetland soil analyzed. Compared with SP3 site, the proportions of Euryarchaeota were decreased by 16.70% and 31.78%, while Thaumarchaeota increased by 7.26% and 17.64% in the SP1 and SP2, respectively. Crenarchaeota was found only in SP3. Shannon–wiener diversity indices in SP1, SP2, and SP3 sites were 3.44, 3.87, and 3.94, respectively, indicating that the diversity of archaea in three plots was: SP3 > SP2 > SP1. Redundancy analysis (RDA) showed that electrical conductivity (EC), soil moisture (SM), hydrogen potential (pH), and soil organic matter content (SOM) may affect archaeal communities. Compared to EC and pH, SM and SOM may have a greater impact on the community composition of archaea.

Characterization and Whole Genome Sequencing of AR23, a Highly Toxic Bacillus thuringiensis Strain Isolated from Lebanese Soil Abstract The demand for sustainable and eco-friendly control methods of pests and insects is increasing worldwide. From this came the interest in Bacillus thuringiensis, an entomopathogenic bacterium capable of replacing chemical pesticides. However, the possibility of pests developing resistance to a particular strain may impair its use, and there is a need to identify novel strains of this species as potential commercial biopesticides. B. thuringiensis sv. israelensis is one of the most successful serovars, widely commercialized for its activity against black fly and mosquito larvae. In this study, we isolated, characterized, and sequenced a new Lebanese B. thuringiensis sv. israelensis isolate, strain AR23. Compared to the commercialized reference strain AM65-52 (Vectobac®, Sumitomo), AR23 showed an increased activity against several mosquito species. The genomic analysis revealed that this strain, compared to AM65-52, possesses a simplified plasmid content and an additional functional cry4Ba coding gene that most likely accounts for the increased effectiveness of this strain in mosquito larvae killing.

Characteristics and Complete Genome Analysis of Bacillus asahii OM18, a Bacterium in Relation to Soil Fertility in Alkaline Soils Under Long-Term Organic Manure Amendment Abstract Bacillus asahii strain OM18, a bacterium in relation to soil fertility, was isolated from alkaline soils under long-term organic manure application in the North China Plain. B. asahii species play a pivotal role in the promotion of both crop yield and soil fertility via accelerating carbon and phosphorus cycling. However, little is known about the characteristics of B. asahii and its underlying molecular mechanism involved in soil nutrient cycling as well as its potential in promoting crop growth. To this end, we report the characteristics and complete genome analysis of strain OM18, which is relevant to promoting plant growth in phosphorus-deficient alkaline soils. Our results provide a glimpse into the metabolic function of B. asahii OM18.

Wickerhamomyces kurtzmanii sp. nov. An Ascomycetous Yeast Isolated From Crater Lake Water, Da Hinggan Ling Mountain, China Abstract One novel ascomycetous yeast strain TF5-16-2 was isolated from water samples of Tuofengling crater lake located in Da Hinggan Ling Mountain, in the Inner Mongolia province of China. Morphological, physiological characteristics, as well as phylogenetic analyses of D1/D2 domains of the large subunit rRNA (LSU), ITS region, small subunit rRNA (SSU), and elongation factor-1α (EF-1α) were performed and finally confirmed the phylogenetic placement of strain TF5-16-2 in the genus Wickerhamomyces. Sequences analysis revealed that strain TF5-16-2 differed from its most closely related phylogenetic neighbors ‘Candida’ silvicultrix CBS 6269T and Wickerhamomyces anomalus CBS 5759T by 8.0% (including 2.3% gaps), 8.5% (including 2.4% gaps) divergences in D1/D2 domains of LSU, and 11% (including 4.3% gaps) and 13% (including 4.4% gaps) divergences in ITS region, respectively. As the considerable sequence divergence and distinguishable physiological characteristics, strain TF5-16-2 was proposed as a new species of the genus Wickerhamomyces, with the name Wickerhamomyces kurtzmanii sp. nov. (holotype = CGMCC 2.5597, Mycobank number is MB829959).