The Fe-S cluster biosynthesis in Enterococcus faecium is essential for anaerobic growth and gastrointestinal colonization.

The facultative anaerobic Gram-positive bacterium Enterococcus faecium is a ubiquitous member of the human gut microbiota. However, it has gradually evolved into a pathogenic and multidrug resistant lineage that causes nosocomial infections. The establishment of high-level intestinal colonization by enterococci represents a critical step of infection. The majority of current research on Enterococcus has been conducted under aerobic conditions, while limited attention has been given to its physiological characteristics in anaerobic environments, which reflects its natural colonization niche in the gut. In this study, a high-density transposon mutant library containing 26,620 distinct insertion sites was constructed. Tn-seq analysis identified six genes that significantly contribute to growth under anaerobic conditions. Under anaerobic conditions, deletion of sufB (encoding Fe-S cluster assembly protein B) results in more extensive and significant impairments on carbohydrate metabolism compared to aerobic conditions. Consistently, the pathways involved in this utilization-restricted carbohydrates were mostly expressed at significantly lower levels in mutant compared to wild-type under anaerobic conditions. Moreover, deletion of sufB or pflA (encoding pyruvate formate lyase-activating protein A) led to failure of gastrointestinal colonization in mice. These findings contribute to our understanding of the mechanisms by which E. faecium maintains proliferation under anaerobic conditions and establishes colonization in the gut.

Simultaneously optimizing multiple properties of ß-glucosidase Bgl6 using combined (semi-)rational design strategies and investigation of the underlying mechanisms.

The performance of ß-glucosidase during cellulose saccharification is determined by thermostability, activity and glucose tolerance. However, conflicts between them make it challenging to simultaneously optimize three properties. In this work, such a case was reported using Bgl6-M3 as a starting point. Firstly, four thermostability-enhancing mutations were obtained using computer-aided engineering strategies (mutant M7). Secondly, substrate binding pocket of M7 was reshaped, generating two mutations that increased activity but decreased glucose tolerance (mutant M9). Then a key region lining active site cavity was redesigned, resulting in three mutations that boosted glucose tolerance and activity. Finally, mutant M12 with simultaneously improved thermostability (half-life of 20-fold), activity (kcat/Km of 5.6-fold) and glucose tolerance (ΔIC50 of 200 mM) was obtained. Mechanisms for property improvement were elucidated by structural analysis and molecular dynamics simulations. Overall, the strategies used here and new insights into the underlying mechanisms may provide guidance for multi-property engineering of other enzymes.

Spatial self-phase modulation of a Gaussian beam transmitted through a ferrofluid.

We report on the experimental observation of the diffraction pattern formed in the far-field region when a high-power continuous-wave laser convergent or divergent Gaussian beam passes through a cuvette with ferrofluid. Two different types of diffraction rings with opposite light-intensity distribution are shown in the far field. The difference between the diffractive patterns is attributed to the interaction of the strong spatial self-phase modulation caused by the refractive index change of the medium with wavefront curvature of the input Gaussian beam. The observed behavior of the diffraction pattern dynamics is interpreted theoretically based on the Fresnel-Kirchhoff integral. The negative polarity of nonlinear refraction can be identified by the central interference profiles and the diffraction pattern. At the same time, the self-defocusing phenomena of the ferrofluid can be determined by the type of pattern. The nonlinear refraction coefficients of the ferrofluid were estimated to be ∼-2.89×10-5cm2/W (convergent Gaussian beam) and ∼-3.53×10-5cm2/W (divergent Gaussian beam). In addition, the corresponding third-order nonlinear optical susceptibility of the sample was also estimated as ∼1.43×10-5esu and ∼1.75×10-5esu, respectively. The experimental results imply a novel potential application of ferrofluid in nonlinear phase modulation devices.

Evolution of Nonlinear Optical Characteristics of Magnetic Nanoparticle Colloidal Suspensions after Laser-Induced Clusters.

In this paper, the nonlinear optical properties of magnetic nanoparticle colloidal solutions were studied by the Z-scan technique using 25 ps laser pulses at a wavelength of 1064 nm. Our results reveal that the formed magnetic nanoparticle clusters under high incident laser intensity will greatly affect the nonlinear optical characteristics of the solution. As the intensity of the pulsed laser decreases, the reverse saturable absorption coefficient and nonlinear refractive index of the sample tend to increase. The evolution of this nonlinear characteristic only occurs in liquid suspension. This is confirmed by fixing particles on a substrate upon which the responses observed in the liquid medium are no longer present. Besides, the possibility of generating optical trapping in the focus of the laser pulses is proposed to explain our experimental results.

Engineering of ß-Glucosidase Bgl15 with Simultaneously Enhanced Glucose Tolerance and Thermostability To Improve Its Performance in High-Solid Cellulose Hydrolysis.

In this study, a Petri-dish-based double-layer high-throughput screening method was established to improve glucose tolerance of ß-glucosidase Bgl15. Two beneficial mutations were identified, and the joint mutant 2R1 improved the half-maximal inhibitory concentration of glucose from 0.04 to 2.1 M. The crystal structure of 2R1 was subsequently determined at 2.7 Å. Structure analysis revealed that enhancement of glucose tolerance may be due to improved transglycosylation activity made possible by a hydrophobic binding site for glucose as an acceptor and more stringent control of a putative water channel. To further ameliorate the application potential of the enzyme, it was engineered to increase the half-life at 50 °C from 0.8 h (Bgl15) to 180 h (mutant 5R1). Furthermore, supplementation of 5R1 to the cellulase cocktail significantly improved glucose production from pretreated sugar cane bagasse by 38%. Consequently, this study provided an efficient approach to enhance glucose tolerance and generated a promising catalyst for cellulose saccharification.

Passively Q-switched Nd:YVO4 laser operating at 1.3 µm with a graphene oxide and ferroferric-oxide nanoparticle hybrid as a saturable absorber.

A self-made saturable absorber (SA) based on hybridized graphene oxide (GO) and ${{\rm Fe}_{3}}{{\rm O}_{4}}$Fe3O4 nanoparticles (FONP) was inserted into a linear cavity to generate a passively $ Q $Q-switched solid-state ${\rm Nd}\text:{{\rm YVO}_4}$Nd:YVO4 laser operating at the 1.3 µm waveband. The laser had a minimum pulse width of 163 ns and a maximum repetition rate of 314 kHz. This experiment, to the best of our knowledge, is the first to demonstrate that hybridized GO and FONP (GO-FONP) can be used as an SA in passively $ Q $Q-switched pulse lasers. Results show that GO-FONP has the potential to be used for passively $ Q $Q-switched laser generation.

Improving Galactooligosaccharide Synthesis Efficiency of ß-Galactosidase Bgal1-3 by Reshaping the Active Site with an Intelligent Hydrophobic Amino Acid Scanning.

There are ongoing interests in improving the galactooligosaccharide (GOS) synthesis efficiency of ß-galactosidase by protein engineering. In this study, an intelligent double-hydrophobic amino acid scanning strategy was proposed and employed to target nine residues forming the glycon-binding site (-1 subsite) of ß-galactosidase Bgal1-3. Two mutants C510V and H512I with significantly improved GOS synthesis efficiency were obtained. When 40% (w/v) lactose was used as a substrate, Bgal1-3 reached a maximum GOS yield of 45.3% at 16 h, while the mutants reached higher yields in a much shorter time (59.1% at 10 h for C510V, 51.5% at 2 h for H512I). When skim milk was treated with these enzymes, more GOS was produced (19.9 g/L for C510V, 12.7 g/L for H512I) than that for Bgal1-3 (10.3 g/L) at a lactose conversion of 90%. These results validated hydrophobicity scanning as an efficient method to engineer ß-galactosidases into promising catalysts for the preparation of GOS and GOS-enriched milk.