Self-assembly of peptide nanofibers with chirality-encoded antimicrobial activity.

The nanostructured antimicrobial agents, self-assembled by the antimicrobial peptides (AMPs), represent an intriguing platform for the treatment of pathogens. Although the structural characteristics significantly influence antimicrobial functionality, the role of chirality is usually ignored and still unclear. Herein, two homochiral AMPs (all L- or all D-amino acids), including C16-LV4LR4 (LL) and C16-DV4DR4 (DD), and a heterochiral AMP with alternating D-/L-amino acids, C16-DV4LR4 (DL), were self-assembled into left-handed, right-handed, and right-handed helical nanofibers, respectively. The valine configuration determined the supramolecular chirality of the nanofibers. However, the DL molecules exhibited a highly aggregated propensity to form more stable helical nanofibers with a lower degree of twist and a larger helical pitch. This characteristic resulted in the optimal antimicrobial activity of the DL nanofibers against both Gram-negative and Gram-positive bacteria. Furthermore, the membrane permeability assay confirmed the higher activity for damaging the cell membrane by the DL nanofibers. These results demonstrated the significance of molecular chirality in directing the self-assembly of the amphiphilic peptides, eventually affecting their antimicrobial activity. This study opens up the possibility to fabricate promising nanostructured antimicrobial materials by controlling the chirality and structure of the materials.

Fabrication of bacterial cellulose with TiO2-ZnO nanocomposites as a multifunctional membrane for water remediation.

Superhydrophilic/underwater superoleophobic (SUS) membrane technology has attracted extensive attention for water purification. However, the fabrication of multifunctional membranes to satisfy the complex wastewater treatment is still a big challenge. In this work, bacterial cellulose (BC) based multifunctional SUS membranes were designed for water purification. Membranes were prepared by blending BC nanofibers with TiO2 nanoparticles (NPs), and further modified by the in situ growth of ZnO-NPs. The composite membranes showed oil/water (o/w) separation under a small driving pressure (0.2-0.3 bar) with a flux rate of 8232.81 ± 212 L m-2h-1 and with a high separation efficiency (>99.9%). Membranes could also separate oil-in-water emulsion with a separation flux of 1498 ± 74 L m-2h-1 and with high efficiency (99.25%). Moreover, the composite membrane exhibited photocatalytic activity under visible light with a high efficiency (>92%). The composite membranes were also investigated for antibacterial activity against Gram-positive and Gram-negative bacterial strains. This work may inspire the fabrication of next-generation multifunctional membranes for wastewater treatment, particularly oily wastewater, dyes and microbial contaminated water.

In situ regulation of bacterial cellulose networks by starch from different sources or amylose/amylopectin content during fermentation.

Bacterial cellulose (BC) is a promising biopolymer, but its three-dimensional structure needs to be controllable to be used in multiple fields. BC has some advantages over other types of cellulose, not only in terms of purity and properties but also in terms of modification (in situ modification) during the synthesis process. Here, starches from different sources or with amylose/amylopectin content were added to the growth medium to regulate the structural properties of BC in-situ. The obtained BC membranes were further modified by superhydrophobic treatment for oil-water separation. Starches alter the viscosity of the medium, thus affecting bacterial motility and cellulose synthesis, and adhere to the microfibers, limiting their further polymerization and ultimately altering the membrane porosity, pore size, and mechanical properties perpendicular to the BC fibril layer direction. The average pore diameter of the BC/PS membrane increased by 1.94 times compared to the initial BC membrane. The chemically modified BC/PS membrane exhibited super-hydrophobicity (water contact angle 167°), high oil-water separation flux (dichloromethane, 23,205 Lm-2 h-1 MPa-1), high separation efficiency (>97%). The study provides a foundation for developing methods to regulate the network structure of BC and broaden its application.

Fabrication of Bacterial Cellulose-Based Dressings for Promoting Infected Wound Healing.

Bacterial cellulose (BC) holds several unique properties such as high water retention capability, flexibility, biocompatibility, and high absorption capacity. All these features make it a potential material for wound healing applications. However, it lacks antibacterial properties, which hampers its applications for infectious wound healings. This study reported BC-based dressings containing ε-polylysine (ε-PL), cross-linked by a biocompatible and mussel-inspired polydopamine (PDA) for promoting infectious wound healing. BC membranes were coated with PDA by a simple self-polymerization process, followed by treating with different contents of ε-PL. The resulted membranes showed strong antibacterial properties against tested bacteria by both in vitro and in vivo evaluations. The membranes also exhibited hemocompatibility and cytocompatibility by in vitro investigations. Moreover, the functionalized membranes promoted infected wound healing using Sprague-Dawley rats as a model animal. A complete wound healing was observed in the group treated with functionalized membranes, while wounds were still open for control and pure BC groups in the same duration. Histological investigations indicated that the thickness of newborn skin was greater and smoother in the groups treated with modified membranes in comparison to neat BC or control groups. These results revealed that the functionalized membranes have great potential as a dressing material for infected wounds in future clinical applications.

[Significance of spinal sagittal balance for guiding acetabular prosthesis positioning].

Since the concept of "safe area" put forward by Lewinnek, it has been widely recognized. While in recent years, many scholars have found that even if the acetabular prosthesis was placed on the "safe area", there were still many unexplained dislocation after total hip arthroplasty. And scholars began to question whether the "safe area" is really suitable for all patients. Spinal degeneration, deformity, lumbar fusion, etc. will lead to spine sagittal imbalance and changes in pelvic activity, which could lead to changes in acetabular orientation, and ultimately lead to edge loading, wear, impact, and even dislocation after total hip replacement. From the perspective of wear, impact and dislocation, it is determined by the functional positioning of the acetabular cup, not the anatomical positioning. The anatomical positioning and functional positioning of the neutral pelvic acetabular cup in the standing position can be considered equivalent. For pelvic rotation more than 20°, functional placement needs to be considered. In recent years, as the understanding of the internal relationship between the spine-pelvis-hip joint has become more and more profound, some scholars further classify the hip-spine relationship according to whether the spine is stiff or deformed, and propose corresponding acetabulums according to different types of hip-spine relationships The function of placement, so as to achieve a stable artificial hip joint. Therefore, it is of great significance to fully assess whether the patient's sagittal plane is balanced before surgery to guide artificial hip replacement surgery.

Sustainable, superhydrophobic membranes based on bacterial cellulose for gravity-driven oil/water separation.

Bacterial cellulose (BC) is a substrate material with high purity and robust mechanical strength, but due to its small pore size and relatively expensive price, it is restricted as an oil-/water separation membrane. In this study, cheaper plant cellulose needle-leaf bleached kraft pulp (NBKP) was added to BC to increase the pore size of the composite membrane, and a superhydrophobic/superoleophilic membrane was prepared for oil-/water separation. The modified membrane surface displayed a petal-like micro-structure and a water contact angle (WCA) of 162.3°, while the oil contact angle was decreased to 0°. What's more, the membrane exhibited excellent oil-/water separation under gravity, recyclability, and a separation efficiency (>95 %), and it was both pH and salt resistant. The membrane also remained durably hydrophobic after 10 separation cycles. And the separation methodology is expected to be highly energy-efficient.

Reusable ternary PVA films containing bacterial cellulose fibers and ε-polylysine with improved mechanical and antibacterial properties.

The combination of high mechanical properties, antibacterial activity and a green synthesis of the polyvinyl alcohol (PVA) based films remains challenging. This study presents a ternary system of PVA films containing bacterial cellulose (BC) and epsilon-polylysine (ε-PL) by a green solution casting method. The prepared composite films showed more than 99% antibacterial properties against both Staphylococcus aureus and Escherichia coli bacteria. Moreover, the films were collected after a single use and were reused twice, which still exhibited strong antibacterial activity. The films showed thermal stability and higher mechanical properties as compared to pure PVA films. In addition, the cytotoxicity of the films was evaluated by MTT assay against NIH 3T3 mouse fibroblast cells. The results showed no toxicity of the films towards tested cells. We believe that these antibacterial films may find applications in active food packaging and biomedical fields.

Thermococcus Eurythermalis Endonuclease IV Can Cleave Various Apurinic/Apyrimidinic Site Analogues in ssDNA and dsDNA.

Endonuclease IV (EndoIV) is a DNA damage-specific endonuclease that mainly hydrolyzes the phosphodiester bond located at 5' of an apurinic/apyrimidinic (AP) site in DNA. EndoIV also possesses 3'-exonuclease activity for removing 3'-blocking groups and normal nucleotides. Here, we report that Thermococcus eurythermalis EndoIV (TeuendoIV) shows AP endonuclease and 3'-exonuclease activities. The effect of AP site structures, positions and clustered patterns on the activity was characterized. The AP endonuclease activity of TeuendoIV can incise DNA 5' to various AP site analogues, including the alkane chain Spacer and polyethylene glycol Spacer. However, the short Spacer C2 strongly inhibits the AP endonuclease activity. The kinetic parameters also support its preference to various AP site analogues. In addition, the efficient cleavage at AP sites requires ≥2 normal nucleotides existing at the 5'-terminus. The 3'-exonuclease activity of TeuendoIV can remove one or more consecutive AP sites at the 3'-terminus. Mutations on the residues for substrate recognition show that binding AP site-containing or complementary strand plays a key role for the hydrolysis of phosphodiester bonds. Our results provide a comprehensive biochemical characterization of the cleavage/removal of AP site analogues and some insight for repairing AP sites in hyperthermophile cells.

The trimeric Hef-associated nuclease HAN is a 3'→5' exonuclease and is probably involved in DNA repair.

Nucleases play important roles in nucleic acid metabolism. Some archaea encode a conserved protein known as Hef-associated nuclease (HAN). In addition to its C-terminal DHH nuclease domain, HAN also has three N-terminal domains, including a DnaJ-Zinc-finger, ribosomal protein S1-like, and oligonucleotide/oligosaccharide-binding fold. To further understand HAN's function, we biochemically characterized the enzymatic properties of HAN from Pyrococcus furiosus (PfuHAN), solved the crystal structure of its DHH nuclease domain, and examined its role in DNA repair. Our results show that PfuHAN is a Mn2+-dependent 3'-exonuclease specific to ssDNA and ssRNA with no activity on blunt and 3'-recessive double-stranded DNA. Domain truncation confirmed that the intrinsic nuclease activity is dependent on the C-terminal DHH nuclease domain. The crystal structure of the DHH nuclease domain adopts a trimeric topology, with each subunit adopting a classical DHH phosphoesterase fold. Yeast two hybrid assay confirmed that the DHH domain interacts with the IDR peptide of Hef nuclease. Knockout of the han gene or its C-terminal DHH nuclease domain in Haloferax volcanii resulted in increased sensitivity to the DNA damage reagent MMS. Our results imply that HAN nuclease might be involved in repairing stalled replication forks in archaea.

NanoRNase from Aeropyrum pernix shows nuclease activity on ssDNA and ssRNA.

In cells, degrading DNA and RNA by various nucleases is very important. These processes are strictly controlled and regulated to maintain DNA integrity and to mature or recycle various RNAs. NanoRNase (Nrn) is a 3'-exonuclease that specifically degrades nanoRNAs shorter than 5 nucleotides. Several Nrns have been identified and characterized in bacteria, mainly in Firmicutes. Archaea often grow in extreme environments and might be subjected to more damage to DNA/RNA, so DNA repair and recycling of damaged RNA are very important in archaea. There is no report on the identification and characterization of Nrn in archaea. Aeropyrum pernix encodes three potential Nrns: NrnA (Ape1437), NrnB (Ape0124), and an Nrn-like protein Ape2190. Biochemical characterization showed that only Ape0124 could degrade ssDNA and ssRNA from the 3'-end in the presence of Mn2+. Interestingly, unlike bacterial Nrns, Ape0124 prefers ssDNA, including short nanoDNA, and degrades nanoRNA with lower efficiency. The 3'-DNA backbone was found to be required for efficiently hydrolyzing the phosphodiester bonds. In addition, Ape0124 also degrads the 3'-overhang of double-stranded DNA. Interestingly, Ape0124 could hydrolyze pAp into AMP, which is a feature of bacterial NrnA, not NrnB. Our results indicate that Ape0124 is a novel Nrn with a combined substrate profile of bacterial NrnA and NrnB.

Two Archaeal RecJ Nucleases from Methanocaldococcus jannaschii Show Reverse Hydrolysis Polarity: Implication to Their Unique Function in Archaea.

Bacterial nuclease RecJ, which exists in almost all bacterial species, specifically degrades single-stranded (ss) DNA in the 5' to 3' direction. Some archaeal phyla, except Crenarchaea, also encode RecJ homologs. Compared with bacterial RecJ, archaeal RecJ exhibits a largely different amino acid sequence and domain organization. Archaeal RecJs from Thermococcus kodakarensis and Pyrococcus furiosus show 5'→3' exonuclease activity on ssDNA. Interestingly, more than one RecJ exists in some Euryarchaeota classes, such as Methanomicrobia, Methanococci, Methanomicrobia, Methanobacteria, and Archaeoglobi. Here we report the biochemical characterization of two RecJs from Methanocaldococcus jannaschii, the long RecJ1 (MJ0977) and short RecJ2 (MJ0831) to understand their enzymatic properties. RecJ1 is a 5'→3' exonuclease with a preference to ssDNA; however, RecJ2 is a 3'→5' exonuclease with a preference to ssRNA. The 5' terminal phosphate promotes RecJ1 activity, but the 3' terminal phosphate inhibits RecJ2 nuclease. Go-Ichi-Ni-San (GINS) complex does not interact with two RecJs and does not promote their nuclease activities. Finally, we discuss the diversity, function, and molecular evolution of RecJ in archaeal taxonomy. Our analyses provide insight into the function and evolution of conserved archaeal RecJ/eukaryotic Cdc45 protein.

Sulfolobus acidocaldarius UDG Can Remove dU from the RNA Backbone: Insight into the Specific Recognition of Uracil Linked with Deoxyribose.

Sulfolobus acidocaldarius encodes family 4 and 5 uracil-DNA glycosylase (UDG). Two recombinant S. acidocaldarius UDGs (SacUDG) were prepared and biochemically characterized using oligonucleotides carrying a deaminated base. Both SacUDGs can remove deoxyuracil (dU) base from both double-stranded DNA and single-stranded DNA. Interestingly, they can remove U linked with deoxyribose from single-stranded RNA backbone, suggesting that the riboses on the backbone have less effect on the recognition of dU and hydrolysis of the C-N glycosidic bond. However, the removal of rU from DNA backbone is inefficient, suggesting strong steric hindrance comes from the 2' hydroxyl of ribose linked to uracil. Both SacUDGs cannot remove 2,2'-anhydro uridine, hypoxanthine, and 7-deazaxanthine from single-stranded DNA and single-stranded DNA. Compared with the family 2 MUG, other family UDGs have an extra N-terminal structure consisting of about 50 residues. Removal of the 46 N-terminal residues of family 5 SacUDG resulted in only a 40% decrease in activity, indicating that the [4Fe-4S] cluster and truncated secondary structure are not the key elements in hydrolyzing the glycosidic bond. Combining our biochemical and structural results with those of other groups, we discussed the UDGs' catalytic mechanism and the possible repair reactions of deaminated bases in prokaryotes.

The crystal structure of Pyrococcus furiosus RecJ implicates it as an ancestor of eukaryotic Cdc45.

RecJ nucleases specifically degrade single-stranded (ss) DNA in the 5' to 3' direction. Archaeal RecJ is different from bacterial RecJ in sequence, domain organization, and substrate specificity. The RecJ from archaea Pyrococcus furiosus (PfuRecJ) also hydrolyzes RNA strands in the 3' to 5' direction. Like eukaryotic Cdc45 protein, archaeal RecJ forms a complex with MCM helicase and GINS. Here, we report the crystal structures of PfuRecJ and the complex of PfuRecJ and two CMPs. PfuRecJ bind one or two divalent metal ions in its crystal structure. A channel consisting of several positively charged residues is identified in the complex structure, and might be responsible for binding substrate ssDNA and/or releasing single nucleotide products. The deletion of the complex interaction domain (CID) increases the values of kcat/Km of 5' exonuclease activity on ssDNA and 3' exonuclease activity on ssRNA by 5- and 4-fold, respectively, indicating that the CID functions as a regulator of enzymatic activity. The DHH domain of PfuRecJ interacts with the C-terminal beta-sheet domain of the GINS51 subunit in the tetrameric GINS complex. The relationship of archaeal and bacterial RecJs, as well as eukaryotic Cdc45, is discussed based on biochemical and structural results.

pSW2, a Novel Low-Temperature-Inducible Gene Expression Vector Based on a Filamentous Phage of the Deep-Sea Bacterium Shewanella piezotolerans WP3.

A low-temperature-inducible protein expression vector (pSW2) based on a filamentous phage (SW1) of the deep-sea bacterium Shewanella piezotolerans WP3 was constructed. This vector replicated stably in Escherichia coli and Shewanella species, and its copy number increased at low temperatures. The pSW2 vector can be utilized as a complementation plasmid in WP3, and it can also be used for the production of complex cytochromes with multiple heme groups, which has the potential for application for metal ion recovery or bioremediation. Promoters of low-temperature-inducible genes in WP3 were fused into the vector to construct a series of vectors for enhancing protein expression at low temperature. The maximum green fluorescent protein intensity was obtained when the promoter for the hfq gene was used. The WP3/pSW2 system can efficiently produce a patatin-like protein (PLP) from a metagenomic library that tends to form inclusion bodies in E. coli. The yields of PLP in the soluble fraction were 8.3 mg/liter and 4.7 mg/liter of culture at 4°C and 20°C, respectively. Moreover, the pSW2 vector can be broadly utilized in other Shewanella species, such as S. oneidensis and S. psychrophila.

Biomineralization mediated by anaerobic methane-consuming cell consortia.

Anaerobic methanotrophic archaea (ANME) play a significant role in global carbon cycles. These organisms consume more than 90% of ocean-derived methane and influence the landscape of the seafloor by stimulating the formation of carbonates. ANME frequently form cell consortia with sulfate-reducing bacteria (SRB) of the family Deltaproteobacteria. We investigated the mechanistic link between ANME and the natural consortium by examining anaerobic oxidation of methane (AOM) metabolism and the deposition of biogenetic minerals through high-resolution imaging analysis. All of the cell consortia found in a sample of marine sediment were encrusted by a thick siliceous envelope consisting of laminated and cementing substances, whereas carbonate minerals were not found attached to cells. Beside SRB cells, other bacteria (such as Betaproteobacteria) were found to link with the consortia by adhering to the siliceous crusts. Given the properties of siliceous minerals, we hypothesize that ANME cell consortia can interact with other microorganisms and their substrates via their siliceous envelope, and this mechanism of silicon accumulation may serve in clay mineral formation in marine sedimentary environments. A mechanism for biomineralization mediated by AOM consortia was suggested based on the above observations.

Methanotrophic archaea possessing diverging methane-oxidizing and electron-transporting pathways.

Anaerobic oxidation of methane (AOM) is a crucial process limiting the flux of methane from marine environments to the atmosphere. The process is thought to be mediated by three groups of uncultivated methane-oxidizing archaea (ANME-1, 2 and 3). Although the responsible microbes have been intensively studied for more than a decade, central mechanistic details remain unresolved. On the basis of an integrated analysis of both environmental metatranscriptome and single-aggregate genome of a highly active AOM enrichment dominated by ANME-2a, we provide evidence for a complete and functioning AOM pathway in ANME-2a. All genes required for performing the seven steps of methanogenesis from CO2 were found present and actively expressed. Meanwhile, genes for energy conservation and electron transportation including those encoding F420H2 dehydrogenase (Fpo), the cytoplasmic and membrane-associated Coenzyme B-Coenzyme M heterodisulfide (CoB-S-SCoM) reductase (HdrABC, HdrDE), cytochrome C and the Rhodobacter nitrogen fixation (Rnf) complex were identified and expressed, whereas genes encoding for hydrogenases were absent. Thus, ANME-2a is likely performing AOM through a complete reversal of methanogenesis from CO2 reduction without involvement of canonical hydrogenase. ANME-2a is demonstrated to possess versatile electron transfer pathways that would provide the organism with more flexibility in substrate utilization and capacity for rapid adjustment to fluctuating environments. This work lays the foundation for understanding the environmental niche differentiation, physiology and evolution of different ANME subgroups.

The regulatory role of ferric uptake regulator (Fur) during anaerobic respiration of Shewanella piezotolerans WP3.

Ferric uptake regulator (Fur) is a global regulator that controls bacterial iron homeostasis. In this study, a fur deletion mutant of the deep-sea bacterium Shewanella piezotolerans WP3 was constructed. Physiological studies revealed that the growth rate of this mutant under aerobic conditions was only slightly lower than that of wild type (WT), but severe growth defects were observed under anaerobic conditions when different electron acceptors (EAs) were provided. Comparative transcriptomic analysis demonstrated that Fur is involved not only in classical iron homeostasis but also in anaerobic respiration. Fur exerted pleiotropic effects on the regulation of anaerobic respiration by controlling anaerobic electron transport, the heme biosynthesis system, and the cytochrome c maturation system. Biochemical assays demonstrated that levels of c-type cytochromes were lower in the fur mutant, consistent with the transcriptional profiling. Transcriptomic analysis and electrophoretic mobility shift assays revealed a primary regulation network for Fur in WP3. These results suggest that Fur may act as a sensor for anoxic conditions to trigger and influence the anaerobic respiratory system.

Abundance and diversity of archaeal accA gene in hot springs in Yunnan Province, China.

It has been suggested that archaea carrying the accA gene, encoding the alpha subunit of the acetyl CoA carboxylase, autotrophically fix CO2 using the 3-hydroxypropionate/4-hydroxybutyrate pathway in low-temperature environments (e.g., soils, oceans). However, little new information has come to light regarding the occurrence of archaeal accA genes in high-temperature ecosystems. In this study, we investigated the abundance and diversity of archaeal accA gene in hot springs in Yunnan Province, China, using DNA- and RNA-based phylogenetic analyses and quantitative polymerase chain reaction. The results showed that archaeal accA genes were present and expressed in the investigated Yunnan hot springs with a wide range of temperatures (66-96 °C) and pH (4.3-9.0). The majority of the amplified archaeal accA gene sequences were affiliated with the ThAOA/HWCG III [thermophilic ammonia-oxidizing archaea (AOA)/hot water crenarchaeotic group III]. The archaeal accA gene abundance was very close to that of AOA amoA gene, encoding the alpha subunit of ammonia monooxygenase. These data suggest that AOA in terrestrial hot springs might acquire energy from ammonia oxidation coupled with CO2 fixation using the 3-hydroxypropionate/4-hydroxybutyrate pathway.