Aside from applications in the production of commercial enzymes and metabolites, Bacillus amyloliquefaciens is also an important group of plant growth-promoting rhizobacteria that supports plant growth and suppresses phytopathogens. A host-genotype-independent counter-selectable marker would enable rapid genetic manipulation and metabolic engineering, accelerating the study of B. amyloliquefaciens and its development as both a microbial cell factory and plant growth-promoting rhizobacteria. Here, a host-genotype-independent counter-selectable marker pheS* was constructed through a point mutation of the gene pheS, which encodes the α-subunit of phenylalanyl-tRNA synthetase in Bacillus subtilis strain 168. In the presence of 5 mM p-chloro-phenylalanine, 100 % of B. amyloliquefaciens strain SQR9 cells carrying pheS* were killed, whereas the wild-type strain SQR9 showed resistance to p-chloro-phenylalanine. A simple pheS* and overlap-PCR-based strategy was developed to create the marker-free deletion of the amyE gene as well as a 37-kb bmy cluster in B. amyloliquefaciens SQR9. The effectiveness of pheS* as a counter-selectable marker in B. amyloliquefaciens was further confirmed through the deletion of amyE genes in strains B. amyloliquefaciens FZB42 and NJN-6. In addition, the potential use of pheS* in other Bacillus species was preliminarily assessed. The expression of PheS* in B. subtilis strain 168 and B. cereus strain ATCC 14579 caused pronounced sensitivity of both hosts to p-chloro-phenylalanine, indicating that pheS* could be used as a counter-selectable marker (CSM) in these strains.

To induce natural genetic competence in Bacillus amyloliquefaciens isolates through overexpression of the master regulator, ComK, from B. subtilis (ComKBsu).Plasmid pUBXC carrying the xylose-inducible comK expression cassette was constructed using plasmid pUB110 as a backbone. Plasmid pUBXC could be transferred from B. subtilis to B. amyloliquefaciens through plasmid pLS20-mediated biparental conjugation. After being induced by xylose, four B. amyloliquefaciens strains harbouring plasmid pUBXC developed genetic competence. Under optimal conditions, the transformation efficiencies of plasmid DNA ranged from 129 ± 20.6 to 1.7 ± 0.1 × 105 cfu (colony-forming units) per μg DNA, and the transformation efficiencies of PCR-assembled deletion constructs ranged from 3.2 ± 0.76 to 3.5 ± 0.42 × 104 cfu per μg DNA in the four tested strains.Artificial induction of genetic competence through overexpressing ComKBsu in B. amyloliquefaciens completed the tasks of replicative plasmid delivery and gene knockout via direct transformation of PCR-generated deletion cassettes.

Strain YF-2T, a Gram-staining-negative, non-motile, non-spore-forming, light-yellow-pigmented bacterium, was isolated from soil samples collected in the city of Yuncheng, Shanxi province of China. Strain YF-2T grew over a temperature range of 25–37 °C, at pH 5.0–8.0 and with 0–5 % (w/v) NaCl. Phylogenetic analysis based on sequence of the 16S rRNA gene showed that strain YF-2T was closely related to strains Flavobacterium akiainvivens CIP 110358T and Flavobacterium hauense KCTC 32147T with 95.99 and 95.92 % sequence similarity, respectively. The dominant fatty acids of strain YF-2T were Summed Feature 3 (comprising C16:1 ω7c and/or C16:1 ω6c) (21.97 %), iso-C15:0 (18.65 %), iso-C17:0 3OH (11.41 %), C16:0 (9.92 %), and anteiso-C15:0 (6.21 %). It contained phosphatidylethanolamine and menaquinone MK-6 as major polar lipid and respiratory quinone, respectively. Strain YF-2T differs from other Flavobacterium species in many characteristics and represents a novel species, for which the name Flavobacterium shanxiense sp. nov. is proposed. The type strain is strain YF-2T (=CCTCC AB 2014079T = JCM 30153T).

A buprofezin-degrading bacterium, YL-1, was isolated from rice field soil. YL-1 was identified as Rhodococcus sp. on the basis of the comparative analysis of 16S rDNA sequences. The strain could use buprofezin as the sole source of carbon and nitrogen for growth and was able to degrade 92.4% of 50 mg L–1 buprofezin within 48 h in liquid culture. During the degradation of buprofezin, four possible metabolites, 2-tert-butylimino-3-isopropyl-1,3,5-thiadiazinan-4-one, N-tert-butyl-thioformimidic acid formylaminomethyl ester, 2-isothiocyanato-2-methyl-propane, and 2-isothiocyanato-propane, were identified using gas chromatography–mass spectrometry (GC–MS) analysis. The catechol 2,3-dioxygenase activity was strongly induced during the degradation of buprofezin. A novel microbial biodegradation pathway for buprofezin was proposed on the basis of these metabolites. The inoculation of soils treated with buprofezin with strain YL-1 resulted in a higher degradation rate than that observed in noninoculated soils, indicating that strain YL-1 has the potential to be used in the bioremediation of buprofezin-contaminated environments.

A novel amidase gene, designated pamh, was cloned from Paracoccus sp. M-1. Site-directed mutagenesis and bioinformatic analysis showed that the PamH protein belonged to the amidase signature enzyme family. PamH was expressed in Escherichia coli, purified, and characterized. The molecular mass of PamH was determined to be 52 kDa with an isoelectric point of 5.13. PamH displayed its highest enzymatic activity at 45°C and at pH 8.0 and was stable within a pH range of 5.0–10.0. The PamH enzyme exhibited amidase activity, aryl acylamidase activity, and acyl transferase activity, allowing it to function across a very broad substrate spectrum. PamH was highly active on aromatic and short-chain aliphatic amides (benzamide and propionamide), moderately active on amino acid amides, and possessed weak urease activity. Of the anilides examined, only propanil was a good substrate for PamH. For propanil, the kcat and Km were 2.8 s−1 and 158 μM, respectively, and the catalytic efficiency value (kcat/Km) was 0.018 μM−1 s−1. In addition, PamH was able to catalyze the acyl transfer reaction to hydroxylamine for both amide and anilide substrates, including acetamide, propanil, and 4-nitroacetanilide; the highest reaction rate was shown with isobutyramide. These characteristics make PamH an excellent candidate for environmental remediation and an important enzyme for the biosynthesis of novel amides.

In our previous study, a phenylurea herbicides degrading strain Sphingobium sp. YBL2 was isolated. In the present study, the degradation pathway of IPU (Isoproturon) in strain YBL2 was investigated by intermediate metabolites identification. HPLC and/or MS/MS analysis showed that, three intermediate metabolites, MDIPU (1-(4-isopropylphenyl)-3-methylurea), DDIPU (1-(4-isopropylphenyl) urea) and 4-IA (4-Isopropylaniline) was produced by strain YBL2. All of these intermediate metabolites could also be efficiently degraded by strain YBL2. In addition, IPU-induced aniline dioxygenase activity and catechol 2,3-dioxygenase activity were detected in strain YBL2. In combination of all these results, we deduced that the metabolic pathway of IPU in strain YBL2 involves two successive N-demethylations, followed by cleavage of the urea side chain and aromatic ring.Highlights► Three metabolites were detected during IPU degradation by strain YBL2. ► Aniline dioxygenase and Catechol 2,3-dioxygenase activity can be induced by IPU. ► IPU and its metabolites can be completely degraded by strain YBL2. ► IPU degradation pathway in strain YBL2 was proposed.