Keratinase can specifically attack disulfide bridges in keratin to convert them from complex to simplified forms. Keratinase thermal stability has drawn attention to various biotechnological industries. In this study, a keratinase DgeKer was identified from a slightly thermophilic species, D. geothermalis. The in silico analysis showed that DgeKer is composed of signal peptide, N-terminal propeptide, mature domain, and C-terminal extension. DgeKer and its C-terminal extension-truncated enzyme (DgeKer-C) were cloned and expressed in E. coli. The purified DgeKer and DgeKer-C showed maximum activity at 70 °C and pH 9–The thermal stability assay (60 °C) showed that the half-life value of DgeKer and DgeKer-C were 103.45 min and 169.10 min, respectively. DgeKer and DgeKer-C were stable at the range of pH from 9 to 11 and showed good tolerance to some metal ions, surfactants and organic solvent. Furthermore, DgeKer could degrade feathers at 70 °C for 60 min. However, the medium became turbid with obvious softening of barbules after being treated with DgeKer-C, which might be due to C-terminal extension. In summary, a thermostable keratinase DgeKer with high efficiency degradation of feathers may have great potential in industry.

Pseudomonas stutzeri A1501, a plant-associated diazotrophic bacterium, prefers to conform to a nitrogen-fixing biofilm state under nitrogen-deficient conditions. The extracytoplasmic function (ECF) sigma factor AlgU is reported to play key roles in exopolysaccharide (EPS) production and biofilm formation in the Pseudomonas genus; however, the function of AlgU in P. stutzeri A1501 is still unclear. In this work, we mainly investigated the role of algU in EPS production, biofilm formation and nitrogenase activity in A1501. The algU mutant ΔalgU showed a dramatic decrease both in the EPS production and the biofilm formation capabilities. In addition, the biofilm-based nitrogenase activity was reduced by 81.4% in the ΔalgU mutant. The transcriptional level of pslA, a key Psl-like (a major EPS in A1501) synthesis-related gene, was almost completely inhibited in the algU mutant and was upregulated by 2.8-fold in the algU-overexpressing strain. A predicted AlgU-binding site was identified in the promoter region of pslA. The DNase I footprinting assays indicated that AlgU could directly bind to the pslA promoter, and β-galactosidase activity analysis further revealed mutations of the AlgU-binding boxes drastically reduced the transcriptional activity of the pslA promoter; moreover, we also demonstrated that AlgU was positively regulated by RpoN at the transcriptional level and negatively regulated by the RNA-binding protein RsmA at the posttranscriptional level. Taken together, these data suggest that AlgU promotes EPS production and nitrogen-fixing biofilm formation by directly activating the transcription of pslA, and the expression of AlgU is controlled by RpoN and RsmA at different regulatory levels.

Cold-adapted esterases are attracting increasing attention owing to their prospective use in biotechnology. In this study, a novel cold-adapted family Ⅳ esterase EstDR4 was identified and obtained from extremophile Deinococcus radiodurans (D. radiodurans). EstDR4 displayed significant substrate preference towards short and medium chain monoesters (C2–C12). It also showed regioselectivity, enantioselectivity and degradation effects on four insecticides. The optimum temperature and pH for EstDR4 activity were 30 °C and pH 8, respectively. Additionally, EstDR4 exhibited relatively high catalytic activity at 0 °C and high stability from 10–40 °C, with over 80% of its initial activity retained after 1 h of incubation. Moreover, EstDR4 activity was stimulated by Tween 80 and Triton X-100, and inhibited by metal ions such as Co²⁺, Cu²⁺ and Zn²⁺ and several organic solvents. Thus, this enzyme shows development potential for many industrial biotechnological applications, including the manufacture of thermolabile pharmaceutical products, cold-wash detergents and insecticide biodegradation.

Flagellar biosynthesis and motility are subject to a four-tiered transcriptional regulatory circuit in Pseudomonas, and the master regulator FleQ appears to be the highest-level regulator in this hierarchical regulatory cascade. Pseudomonas stutzeri A1501 is motile by a polar flagellum; however, the motility and regulatory mechanisms involved in this process are unknown. Here, we searched the A1501 genome for flagella and motility genes and found that approximately 50 genes, which were distributed in three non-contiguous chromosomal regions, contribute to the formation, regulation and function of the flagella. The non-polar mutation of fleQ impaired flagellar biosynthesis, motility and root colonization but enhanced biofilm formation. FleQ positively regulates the expression of flagellar class II–IV genes, suggesting a regulatory cascade that is coordinated similar to that of the well-known P. aeruginosa. Based on our results, we propose that flagellar genes in P. stutzeri A1501 are regulated in a cascade regulated by FleQ and that flagellum-driven motility properties may be necessary for competitive rhizosphere colonization.

EPSP synthase is the target enzyme of glyphosate herbicides. Due to the extensive use of glyphosate, it is very important to obtain EPSPS genes with high glyphosate resistance for the development of transgenic crops. GR79-EPSPS is a class I EPSP synthase with certain glyphosate resistance isolated from glyphosate-contaminated soil. After more than 1000 generations, a Y40I substitution was identified, and the enzyme had a nearly 1.8-fold decrease in Km [PEP] and a 1.7-fold increase in Ki[glyphosate] compared to the wild-type enzyme. Enzyme dynamics and molecular dynamics analysis showed that the substitution was near the hinge region of EPSPS, and the affinity of glyphosate binding to amino acid residues of the active site decreased due to Y40I substitution, resulting in an increase in glyphosate resistance. These results provide more evidence for the combination of directed evolution and rational design of protein engineering.

Noncoding RNAs (ncRNAs) can finely control the expression of target genes at the posttranscriptional level in prokaryotes. Regulatory small RNAs (sRNAs) designed to control target gene expression for applications in metabolic engineering and synthetic biology have been successfully developed and used. However, the effect on the heterologous expression of species- or strain-specific ncRNAs in other bacterial strains remains poorly understood. In this work, aPseudomonas stutzerispecies-specific regulatory ncRNA, NfiS, which has been shown to play an important role in the response to oxidative stress as well as osmotic stress inP. stutzeriA1501, was cloned and transferred to theEscherichia colistrain Trans10. Recombinant NfiS-expressingE. coli, namely, Trans10-nfiS, exhibited significant enhancement of tolerance to oxidative stress. To map the possible gene regulatory networks mediated by NfiS inE. coliunder oxidative stress, a microarray assay was performed to delineate the transcriptomic differences between Trans10-nfiSand wild-typeE. coliunder H₂O₂shock treatment conditions. In all, 1184 genes were found to be significantly altered, and these genes were divided into mainly five functional categories: stress response, regulation, metabolism related, transport or membrane protein and unknown function. Our results suggest that theP. stutzerispecies-specific ncRNA NfiS acts as a regulator that integrates adaptation to H₂O₂with other cellular stress responses and helps protectE. colicells against oxidative damage.