As Cas9-mediated cleavage requires both protospacer and protospacer adjacent motif (PAM) sequences, it is impossible to employ the CRISPR/Cas9 system to directly edit genomic sites without available PAM sequences nearby. Here, we optimized the CRISPR/Cas9 system and developed an innovative two-step strategy for efficient genome editing of any sites, which did not rely on the availability of PAM sequences. An antibiotic resistance cassette was employed as both a positive and a negative selection marker. By integrating the optimized two-plasmid CRISPR/Cas system and donor DNA, we achieved gene insertion and point mutation with high efficiency in Escherichia coli, and importantly, obtained clean mutants with no other unwanted mutations. Moreover, genome editing of essential genes was successfully achieved using this approach with a few modifications. Therefore, our newly-developed method is PAM-independent and can be used to edit any genomic loci, and we hope this method can also be used for efficient genome editing in other organisms.

A halophilic archaeon named strain LR21^T was isolated from a salt mine in Yunnan Province, PR China. Cells were spherical, Gram-stain-negative and motile. Strain LR21^T grew at 20–50 °C (optimum, 42 °C), with 8–30 % (w/v) NaCl (optimum, 23 %) and at pH 5.5–9.5 (optimum, pH 7.5–8.5). Mg²⁺ was not required for growth. The major polar lipid profile comprised phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and phosphatidylglycerol sulfate. Strain LR21^T had two dissimilar 16S rRNA genes (rrnA and rrnB) and they were closely related to Halomicroarcula limicola YGHS32^T, Hma. pellucida BNERC31^T and Hma. salina YGHS18^T with sequence similarities of 95.3–99.0, 93.0–96.2 and 93.2–95.9 %, respectively, and much lower values to other members. The rpoB′ gene sequence similarities between strain LR21^T and Hma. limicola YGHS32^T, Hma. pellucida BNERC31^T and Hma. salina YGHS18^T were 95.2, 91.2 and 91.2 % respectively. The values of average nucleotide identity (ANI) and average amino-acid identity (AAI) between strain LR21^T and Hma. limicola YGHS32^T, were 89.0 and 90.1 %, respectively. DNA relatedness between strains LR21^T and Hma. limicola YGHS32^T determined by in silico DNA–DNA hybridization was 36.8 %. Values of ANI and AAI between strain LR21^T and other members in the genus Halomicroarcula were far below 95 % and the DNA–DNA relatedness values between strain LR21^T and its close relatives were much lower than 70 %, which is far below the boundary for delineation of a new species prokaryote. The DNA G+C content of strain LR21^T was 62.0 mol% (genome). The results suggested that strain LR21^T represents a novel species of the genus Halomicroarcula , for which the name Halomicroarcula amylolytica sp. nov. is proposed. The type strain is LR21^T (=CGMCC 1.13611^T=NBRC 113588^T).

Two novel extremely halophilic archaeal strains, designated H27T and FL145T, were isolated from a salt mine and a kelp salt sample, respectively. Cells of both strains were Gram-stain-negative, motile and pleomorphic. The 16S rRNA and rpoB′ gene sequence similarities between strains H27T and FL145T were 96.60 and 88.77%. Strains H27T and FL145T were both closely related to Halorhabdus rudnickae WSM-64T, Halorhabdus tiamatea SARL4BT and Halorhabdus utahensis AX-2T, with a 16S rRNA gene sequence similarities of 98.14, 96.34 and 96.27% for strain H27T and 96.42, 95.82 and 96.17% for strain FL145T. The genome-based average nucleotide identity (ANI) values between strains H27T and FL145T, and these three species were 83.93, 79.79 and 79.09% (for strain H27T), and 78.32, 77.95 and 77.05% (for strain FL145T), respectively. The ANI value between strains H27T and FL145T was 78.65 %. The digital DNA–DNA hybridization values between strains H27T and FL145T, and these three species were less than 27.40%, which were below the recommended threshold for membership of the same species. The major polar lipids of both strains were found to consist of sulfated diglycosyl diether, triglycosyl diether, phosphatidylglycerol phosphate methyl ester and phosphatidylglycerol. The DNA G+C content was determined from genome to be 62.10 mol% for strain H27T and 61.51 mol% for strain FL145T. Based on phylogenetic, phenotypic, chemotaxonomic and genomic analyses, these two new isolates should be classified as representing two novel species in the genus Halorhabdus , with strain H27T (=CGMCC 1.16342T=NBRC 113589T) as the type strain of a new species for which we propose the name Halorhabdus amylolytica sp. nov., and strain FL145T (=CGMCC 1.13888T=NBRC 114260T) as the type strain of another new species for which we propose the name Halorhabdus salina sp. nov.

Phthalates esters (PAEs) are a kind of polymeric material additives widely been added into plastics to improve products’ flexibility. It can easily cause environmental pollution which are hazards to public health. In this study, we isolated an efficient PAEs degrading strain, Janthinobacterium sp. E1, and determined its degradation effect of di-2-ethylhexyl phthalate (DEHP) under stress conditions. Strain E1 showed an obvious advantage in pollutants degradation under various environmental stress conditions. Degradation halo clearly occurred around the colony of strain E1 on agar plate supplemented with triglyceride. Strain E1’s esterase is a constitutively expressed intracellular enzyme. The esterase purified from strain E1 showed a higher catalytic effect on short-chain PAEs than long-chain PAEs. The input of DEHP, DBP (dibutyl phthalate) and DMP (dimethyl phthalate) into the tested soil did not change the species composition of soil prokaryotic community, but altered the dominant species in specific environmental conditions. And the community diversity and richness decreased to a certain extent. However, the diversity and richness of the microbial community were improved after the contaminated soil was treated with the strain E1. Our results also suggested that strain E1 exhibited a tremendous potential in environmental bioremediation in the real environment, which provides a new insight into the elimination of the pollutants contamination in the urban environment.

Large Ag dendrites were rapidly deposited via a facile electrochemical route at a deposition current of 20 mA for 10 min in air at room temperature, employing a mixed system of AgNO₃ and NH₃·H₂O as the electrolyte. The phase and morphology of the as-prepared product were characterized by means of powder X-ray diffraction (XRD), energy dispersive spectrometry (EDS), transmission electron microscopy (TEM), selected area electron diffraction (SAED) and scanning electron microscopy (SEM). It was found that the original ammonia amount played a crucial role in the formation of Ag dendrites. Experiments showed that the as-deposited Ag dendrites exhibited versatile applications in some fields including super-SERS performance for the detection of rhodamine-6G, highly effective antimicrobial activities toward Escherichia coli and excellent catalytic capacities for the reduction of 4-nitrophenol to 4-aminophenol.