Films based on TEMPO-oxidized chitosan nanoparticles: Obtaining and potential application as wound dressings.

A series of novel films based on TEMPO-oxidized chitosan nanoparticles were prepared by casting method. Fourier transform infrared spectroscopy (FTIR) was employed to ascertain the chemical structure of TEMPO-oxidized chitosan. The surface morphology of the TEMPO-oxidized chitosan nanoparticles was analyzed by atomic force microscopy (AFM). The physicochemical (area density, thickness, iodine sorption, roughness), functional (moisture sorption, liquid absorption capacity, weight loss upon contact with the liquid, and water vapor transmission rate), antibacterial, and antioxidant properties of films based on TEMPO-oxidized chitosan nanoparticles were also investigated. The physicochemical properties of the films varied widely: area density ranged from 77.83 ± 0.06 to184.46 ± 0.05 mg/cm2, thickness varied between 80.5 ± 1.6 and 200.5 ± 1.6 µm, iodine sorption spanned from 333.7 ± 2.1 to166.4 ± 2.2 mg I2/g, and roughness ranged from 4.1 ± 0.2 to 5.6 ± 0.3 nm. Similarly, the functional properties also varied significantly: moisture sorption ranged from 4.76 ± 0.03 to 9.62 ± 0.11 %, liquid absorption capacity was between 129.04 ± 0.24 and 159.33 ± 0.73 % after 24 h, weight loss upon contact with the liquid varied between 31.06 ± 0.35 and 45.88 ± 0.58 % after 24 h and water vapor transmission rate ranged from 1220.10 ± 2.91to1407.77 ± 5.22 g/m2 day. Despite the wide variations in physicochemical and functional properties, all films showed maximum bacterial reduction of Staphylococcus aureus and Escherichia coli, although they exhibited low antioxidant activity. The results suggest that the films could be effectively utilized as antibacterial wound dressings.

Investigating and Engineering an 1,2-Propanediol-Responsive Transcription Factor-Based Biosensor.

Transcription factor (TF)-based biosensors have arisen as powerful tools in the advancement of metabolic engineering. However, with the emergence of numerous bioproduction targets, the variety of applicable TF-based biosensors remains severely limited. In this study, we investigated and engineered an 1,2-propanediol (1,2-PD)-responsive transcription activator, PocR, from Salmonella typhimurium to enrich the current biosensor repertoire. Heterologous characterization of PocR in E. coli revealed a significantly limited operational range and dynamic range, primarily attributed to the leaky binding between PocR and its corresponding promoters in the absence of the 1,2-PD inducer. Promiscuity characterization uncovered the minor responsiveness of PocR toward glycerol and 1,2-butanediol (1,2-BD). Using AlphaFold-predicted structure and protein mutagenesis, we preliminarily explored the underlying mechanism of PocR. Based on the investigated mechanism, we engineered a PcoR-F46R/G105D variant with an altered inducer specificity to glycerol, as well as a PocR-ARE (Q107A/S192R/A203E) variant with nearly a 4-fold higher dynamic range (6.7-fold activation) and a 20-fold wider operational range (0-20 mM 1,2-PD). Finally, we successfully converted PocR to a repressor through promoter engineering. Integrating the activation and repression functions established a versatile 1,2-PD-induced bifunctional regulation system based on PocR-ARE. Our work showcases the exploration and exploitation of an underexplored type of transcriptional activator capable of recruiting RNA polymerase. It also expands the biosensor toolbox by providing a 1,2-PD-responsive bifunctional regulator and glycerol-responsive activator.

Tyrosol blocks E. coli anaerobic biofilm formation via YbfA and FNR to increase antibiotic susceptibility.

Bacteria within mature biofilms are highly resistant to antibiotics than planktonic cells. Oxygen limitation contributes to antibiotic resistance in mature biofilms. Nitric oxide (NO) induces biofilm dispersal; however, low NO levels stimulate biofilm formation, an underexplored process. Here, we introduce a mechanism of anaerobic biofilm formation by investigating the antibiofilm activity of tyrosol, a component in wine. Tyrosol inhibits E. coli and Pseudomonas aeruginosa biofilm formation by enhancing NO production. YbfA is identified as a target of tyrosol and its downstream targets are sequentially determined. YbfA activates YfeR, which then suppresses the anaerobic regulator FNR. This suppression leads to decreased NO production, elevated bis-(3'-5')-cyclic dimeric GMP levels, and finally stimulates anaerobic biofilm formation in the mature stage. Blocking YbfA with tyrosol treatment renders biofilm cells as susceptible to antibiotics as planktonic cells. Thus, this study presents YbfA as a promising antibiofilm target to address antibiotic resistance posed by biofilm-forming bacteria, with tyrosol acting as an inhibitor.

Impact of the COVID-19 pandemic on extended-spectrum ß-lactamase producing Escherichia coli in urinary tract and blood stream infections: results from a nationwide surveillance network, Finland, 2018 to 2022.

BACKGROUND: Before the COVID-19 pandemic there has been a constant increase in antimicrobial resistance (AMR) of Escherichia coli, the most common cause of urinary tract infections and bloodstream infections. The aim of this study was to investigate the impact of the COVID-19 pandemic on extended-spectrum ß-lactamase (ESBL) production in urine and blood E. coli isolates in Finland to improve our understanding on the source attribution of this major multidrug-resistant pathogen. METHODS: Susceptibility test results of 564,233 urine (88.3% from females) and 23,860 blood E. coli isolates (58.8% from females) were obtained from the nationwide surveillance database of Finnish clinical microbiology laboratories. Susceptibility testing was performed according to EUCAST guidelines. We compared ESBL-producing E. coli proportions and incidence before (2018-2019), during (2020-2021), and after (2022) the pandemic and stratified these by age groups and sex. RESULTS: The annual number of urine E. coli isolates tested for antimicrobial susceptibility decreased 23.3% during 2018-2022 whereas the number of blood E. coli isolates increased 1.1%. The annual proportion of ESBL-producing E. coli in urine E. coli isolates decreased 28.7% among males, from 6.9% (average during 2018-2019) to 4.9% in 2022, and 28.7% among females, from 3.0 to 2.1%. In blood E. coli isolates, the proportion decreased 32.9% among males, from 9.3 to 6.2%, and 26.6% among females, from 6.2 to 4.6%. A significant decreasing trend was also observed in most age groups, but risk remained highest among persons aged ≥ 60 years. CONCLUSIONS: The reduction in the proportions of ESBL-producing E. coli was comprehensive, covering both specimen types, both sexes, and all age groups, showing that the continuously increasing trends could be reversed. Decrease in international travel and antimicrobial use were likely behind this reduction, suggesting that informing travellers about the risk of multidrug-resistant bacteria, hygiene measures, and appropriate antimicrobial use is crucial in prevention. Evaluation of infection control measures in healthcare settings could be beneficial, especially in long-term care.

An efficient ratiometric fluorescence and colorimetric dual-mode probe for convenient determination of nitrite in real samples and E. coli.

Developing an effective and convenient nitrite detection method is indispensable in food safety, environmental monitoring, clinical diagnosis of diseases, and many other areas. Herein, a dicyanoisophorone derivative, TMN-NH2 with large Stokes shift and near-infrared (NIR) emission, was proposed as a ratiometric fluorescence and colorimetric dual-mode probe for the rapid determination of NO2- in acidic media, showing excellent selectivity and high sensitivity. The sensing mechanism is based on the diazotization of TMN-NH2 with NO2- and subsequent diazonium salt hydrolysis to form a hydroxyl-substituted product (TMN-OH). Under the optimized conditions of reaction and detection, a new quantitative analysis method based on TMN-NH2 was established for NO2- detection, exhibiting good linear relationships to NO2- in the range of 0.5 to 15 µM with practical detection limits of 26.6 nM and 17.6 nM for the colorimetric and fluorescent readout, respectively. The quantitative detection of NO2- in real samples demonstrated satisfactory recoveries and repeatability. Moreover, TMN-NH2 was successfully applied for monitoring NO2- in Escherichia coli by confocal fluorescence imaging.

AutoBioTech─A Versatile Biofoundry for Automated Strain Engineering.

The inevitable transition from petrochemical production processes to renewable alternatives has sparked the emergence of biofoundries in recent years. Manual engineering of microbes will not be sufficient to meet the ever-increasing demand for novel producer strains. Here we describe the AutoBioTech platform, a fully automated laboratory system with 14 devices to perform operations for strain construction without human interaction. Using modular workflows, this platform enables automated transformations of Escherichia coli with plasmids assembled via modular cloning. A CRISPR/Cas9 toolbox compatible with existing modular cloning frameworks allows automated and flexible genome editing of E. coli. In addition, novel workflows have been established for the fully automated transformation of the Gram-positive model organism Corynebacterium glutamicum by conjugation and electroporation, with the latter proving to be the more robust technique. Overall, the AutoBioTech platform excels at versatility due to the modularity of workflows and seamless transitions between modules. This will accelerate strain engineering of Gram-negative and Gram-positive bacteria.

Directed Evolution of Acoustic Reporter Genes Using High-Throughput Acoustic Screening.

A major challenge in the fields of biological imaging and synthetic biology is noninvasively visualizing the functions of natural and engineered cells inside opaque samples such as living animals. One promising technology that addresses this limitation is ultrasound (US), with its penetration depth of several cm and spatial resolution on the order of 100 µm. Within the past decade, reporter genes for US have been introduced and engineered to link cellular functions to US signals via heterologous expression in commensal bacteria and mammalian cells. These acoustic reporter genes (ARGs) represent a novel class of genetically encoded US contrast agent, and are based on air-filled protein nanostructures called gas vesicles (GVs). Just as the discovery of fluorescent proteins was followed by the improvement and diversification of their optical properties through directed evolution, here we describe the evolution of GVs as acoustic reporters. To accomplish this task, we establish high-throughput, semiautomated acoustic screening of ARGs in bacterial cultures and use it to screen mutant libraries for variants with increased nonlinear US scattering. Starting with scanning site saturation libraries for two homologues of the primary GV structural protein, GvpA/B, two rounds of evolution resulted in GV variants with 5- and 14-fold stronger acoustic signals than the parent proteins. We anticipate that this and similar approaches will help high-throughput protein engineering play as large a role in the development of acoustic biomolecules as it has for their fluorescent counterparts.

Escherichia coli DNA replication: the old model organism still holds many surprises.

Research on Escherichia coli DNA replication paved the groundwork for many breakthrough discoveries with important implications for our understanding of human molecular biology, due to the high level of conservation of key molecular processes involved. To this day, it attracts a lot of attention, partially by virtue of being an important model organism, but also because the understanding of factors influencing replication fidelity might be important for studies on the emergence of antibiotic resistance. Importantly, the wide access to high-resolution single-molecule and live-cell imaging, whole genome sequencing, and cryo-electron microscopy techniques, which were greatly popularized in the last decade, allows us to revisit certain assumptions about the replisomes and offers very detailed insight into how they work. For many parts of the replisome, step-by-step mechanisms have been reconstituted, and some new players identified. This review summarizes the latest developments in the area, focusing on (a) the structure of the replisome and mechanisms of action of its components, (b) organization of replisome transactions and repair, (c) replisome dynamics, and (d) factors influencing the base and sugar fidelity of DNA synthesis.

IbpAB small heat shock proteins are not host factors for bacteriophage ϕX174 replication.

Bacteriophage ϕX174 is a small icosahedral virus of the Microviridae with a rapid replication cycle. Previously, we found that in ϕX174 infections of Escherichia coli, the most highly upregulated host proteins are two small heat shock proteins, IbpA and IbpB, belonging to the HSP20 family, which is a universally conserved group of stress-induced molecular chaperones that prevent irreversible aggregation of proteins. Heat shock proteins were found to protect against ϕX174 lysis, but IbpA/B have not been studied. In this work, we disrupted the ibpA and ibpB genes and measured the effects on ϕX174 replication. We found that in contrast to other E. coli heat shock proteins, they are not necessary for ϕX174 replication; moreover, their absence has no discernible effect on ϕX174 fecundity. These results suggest IbpA/B upregulation is a response to ϕX174 protein expression but does not play a role in phage replication, and they are not Microviridae host factors.

Analysis of Fluorescent Proteins for Observing Single Gene Locus in a Live and Fixed Escherichia coli Cell.

Fluorescent proteins (FPs) are essential tools for advanced microscopy techniques such as super-resolution imaging, single-particle tracking, and quantitative single-molecule counting. Various FPs fused to DNA-binding proteins have been used to observe the subcellular location and movement of specific gene loci in living and fixed bacterial cells. However, quantitative assessments of the properties of FPs for gene locus measurements are still lacking. Here, we assessed various FPs to observe specific gene loci in live and fixed Escherichia coli cells using a fluorescent repressor-operator binding system (FROS), tet operator-Tet repressor proteins (TetR). Tsr-fused FPs were used to assess the intensity and photostability of various FPs (five red FPs: mCherry2, FusionRed, mRFP, mCrimson3, and dKatushka; and seven yellow FPs: SYFP2, Venus, mCitrine, YPet, mClover3, mTopaz, and EYFP) at the single-molecule level in living cells. These FPs were then used for gene locus measurements using FROS. Our results indicate that TetR-mCrimson3 (red) and TetR-EYFP (yellow) had better properties for visualizing gene loci than the other TetR-FPs. Furthermore, fixation procedures affected the clustering of diffusing TetR-FPs and altered the locations of the TetR-FP foci. Fixation with formaldehyde consistently disrupted proper DNA locus observations using TetR-FPs. Notably, the foci measured using TetR-mCrimson3 remained close to their original positions in live cells after glyoxal fixation. This in vivo study provides a cell-imaging guide for the use of FPs for gene-locus observation in E. coli and a scheme for evaluating the use of FPs for other cell-imaging purposes.

Evaluation of the Antibacterial, Anti-Inflammatory, And Bone-Promoting Capacity of UiO-66 Loaded with Thymol or Carvacrol.

Oral infectious diseases have a significant impact on the health of oral and maxillofacial regions, as well as the overall well-being of individuals. Carvacrol and thymol, two isomers known for their effective antibacterial and anti-inflammatory properties, have gained considerable attention in the treatment of oral infectious diseases. However, their application as topical drugs for oral use is limited due to their poor physical and chemical stability. UiO-66, a metal-organic framework based on zirconium ion (Zr4+), exhibits high drug loading capability. Carvacrol and thymol were efficiently loaded onto UiO-66 with loading rates of 79.60 ± 0.71% and 79.65 ± 0.76%, respectively. The release rates of carvacrol and thymol were 77.82 ± 0.87% and 76.51 ± 0.58%, respectively, after a period of 72 h. Moreover, Car@UiO-66 and Thy@UiO-66 demonstrated excellent antibacterial properties against Candida albicans, Escherichia coli, and Staphylococcus aureus with minimum bactericidal concentrations (MBC) of 0.313 mg/mL, 0.313 mg/mL, and 1.25 mg/mL, respectively. Furthermore, based on the results of the CCK8 cytotoxicity assay, even at concentrations as high as 1.25 mg/mL, Car@UiO-66 and Thy@UiO-66 exhibited excellent biocompatibility with a relative cell survival rate above 50%. These findings suggest that Car@UiO-66 and Thy@UiO-66 possess favorable biocompatibility properties without significant toxicity towards periodontal membrane cells. Additionally, in vivo studies confirmed the efficacy of Car@UiO-66and Thy@UiO-66 in reducing inflammation, promoting bone formation through inhibition of TNF-a and IL6 expression, enhancement of IL10 expression, and acceleration of bone defect healing. Therefore, the unique combination of antibacterial, anti-inflammatory, and osteogenic properties make Car@UiO-66 and Thy@Ui O-66 promising candidates for the treatment of oral infectious diseases and repairing bone defects.

Silver peroxide-incorporated carbon dots with high photothermal performance for combating bacteria.

The widespread use of antibiotics often increases bacterial resistance. Herein, we reported a silver peroxide-incorporated carbon dots (defined as Ag2O2-CDs) with high photothermal conversion efficiency viain situoxidation process. The prepared Ag2O2-CDs exhibited ultra-small size of 2.0 nm and hybrid phase structure. Meanwhile, the Ag2O2-CDs were of a similar optical performance comparing with traditional carbon dots (CDs). Importantly, the incorporation of Ag2O2into CDs significantly enhanced photothermal conversion efficiency from 3.8% to 28.5%. By combining silver ion toxicity and photothermal ablation, the Ag2O2-CDs were capable of destroying gram-positive and gram-negative bacterium effectively. These findings demonstrated that the Ag2O2-CDs could be served as a potential antibacterial agent for clinical applications.

Native mass spectrometry and structural studies reveal modulation of MsbA-nucleotide interactions by lipids.

The ATP-binding cassette (ABC) transporter, MsbA, plays a pivotal role in lipopolysaccharide (LPS) biogenesis by facilitating the transport of the LPS precursor lipooligosaccharide (LOS) from the cytoplasmic to the periplasmic leaflet of the inner membrane. Despite multiple studies shedding light on MsbA, the role of lipids in modulating MsbA-nucleotide interactions remains poorly understood. Here we use native mass spectrometry (MS) to investigate and resolve nucleotide and lipid binding to MsbA, demonstrating that the transporter has a higher affinity for adenosine 5'-diphosphate (ADP). Moreover, native MS shows the LPS-precursor 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo)2-lipid A (KDL) can tune the selectivity of MsbA for adenosine 5'-triphosphate (ATP) over ADP. Guided by these studies, four open, inward-facing structures of MsbA are determined that vary in their openness. We also report a 2.7 Å-resolution structure of MsbA in an open, outward-facing conformation that is not only bound to KDL at the exterior site, but with the nucleotide binding domains (NBDs) adopting a distinct nucleotide-free structure. The results obtained from this study offer valuable insight and snapshots of MsbA during the transport cycle.

Radiolytic Effects on Biological and Abiotic Amino Acids in Shallow Subsurface Ices on Europa and Enceladus.

Europa and Enceladus are key targets to search for evidence of life in our solar system. However, the surface and shallow subsurface of both airless icy moons are constantly bombarded by ionizing radiation that could degrade chemical biosignatures. Therefore, sampling of icy surfaces in future life detection missions to Europa and Enceladus requires a clear understanding of the necessary ice depth where unaltered organic biomolecules might be present. We conducted radiolysis experiments by exposing individual amino acids in ices and amino acids from dead microorganisms in ices to gamma radiation to simulate conditions on these icy worlds. In the pure amino acid samples, glycine did not show a detectable decrease in abundance, whereas the abundance of isovaline decreased by 40% after 4 MGy of exposure. Amino acids in dead Escherichia coli (E. coli) organic matter exhibited a gradual decline in abundances with the increase of exposure dosage, although at much slower rates than individual amino acids. The majority of amino acids in dead A. woodii samples demonstrated a step function decline as opposed to a gradual decline. After the initial drop in abundance with 1 MGy of exposure, those amino acids did not display further decreases in abundance after exposure up to 4 MGy. New radiolysis constants for isolated amino acids and amino acids in dead E. coli material for Europa/Enceladus-like conditions have been derived. Slow rates of amino acid destruction in biological samples under Europa and Enceladus-like surface conditions bolster the case for future life detection measurements by Europa and Enceladus lander missions. Based on our measurements, the "safe" sampling depth on Europa is ∼20 cm at high latitudes of the trailing hemisphere in the area of little impact gardening. Subsurface sampling is not required for the detection of amino acids on Enceladus-these molecules will survive radiolysis at any location on the Enceladus surface. If the stability of amino acids observed in A. woodii organic materials is confirmed in other microorganisms, then the survival of amino acids from a potential biosphere in Europa ice would be significantly increased.

Facile Preparation of Tough, Puncture-Resistant Antibacterial Polyrotaxane Hydrogel.

Slide-ring hydrogels containing polyrotaxane structures have been widely developed, but current methods are more complex, in which modified cyclodextrins, capped polyrotaxanes, and multistep reactions are often needed. Here, a simple one-pot method dissolving the pseudopolyrotaxane (pPRX) in a mixture of acrylamide and boric acid to form a slide-ring hydrogel by UV light is used to construct a tough, puncture-resistant antibacterial polyrotaxane hydrogel. As a new dynamic ring cross-linking agent, boric acid effectively improves the mechanical properties of the hydrogel and involves the hydrogel with fracture toughness. The polyrotaxane hydrogel can withstand 1 MPa compression stress and maintain the morphology integrity, showing 197.5 mJ puncture energy under a sharp steel needle puncture. Meanwhile, its significant antibacterial properties endow the hydrogel with potential applications in the biomedical field.

Unlocking Wearable Microbial Fuel Cells for Advanced Wound Infection Treatment.

Better infection control will accelerate wound healing and alleviate associated healthcare burdens. Traditional antibacterial dressings often inadequately control infections, inadvertently promoting antibacterial resistance. Our research unveils a novel, dual-functional living dressing that autonomously generates antibacterial agents and delivers electrical stimulation, harnessing the power of spore-forming Bacillus subtilis. This dressing is built on an innovative wearable microbial fuel cell (MFC) framework, using B. subtilis endospores as a powerful, dormant biocatalyst. The endospores are resilient, reactivating in nutrient-rich wound exudate to produce electricity and antibacterial compounds. The combination allows B. subtilis to outcompete pathogens for food and other resources, thus fighting infections. The strategy is enhanced by the extracellular synthesis of tin oxide and copper oxide nanoparticles on the endospore surface, boosting antibacterial action, and electrical stimulation. Moreover, the MFC framework introduces a pioneering dressing design featuring a conductive hydrogel embedded within a paper-based substrate. The arrangement ensures cell stability and sustains a healing-friendly moist environment. Our approach has proven very effective against three key pathogens in biofilms: Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus demonstrating exceptional capabilities in both in vitro and ex vivo models. Our innovation marks a significant leap forward in wearable MFC-based wound care, offering a potent solution for treating infected wounds.

Study on the Antibacterial Activity and Bone Inductivity of Nanosilver/PLGA-Coated TI-CU Implants.

Background: Implants are widely used in the field of orthopedics and dental sciences. Titanium (TI) and its alloys have become the most widely used implant materials, but implant-associated infection remains a common and serious complication after implant surgery. In addition, titanium exhibits biological inertness, which prevents implants and bone tissue from binding strongly and may cause implants to loosen and fall out. Therefore, preventing implant infection and improving their bone induction ability are important goals. Purpose: To study the antibacterial activity and bone induction ability of titanium-copper alloy implants coated with nanosilver/poly (lactic-co-glycolic acid) (NSPTICU) and provide a new approach for inhibiting implant-associated infection and promoting bone integration. Methods: We first examined the in vitro osteogenic ability of NSPTICU implants by studying the proliferation and differentiation of MC3T3-E1 cells. Furthermore, the ability of NSPTICU implants to induce osteogenic activity in SD rats was studied by micro-computed tomography (micro-CT), hematoxylin-eosin (HE) staining, masson staining, immunohistochemistry and van gieson (VG) staining. The antibacterial activity of NSPTICU in vitro was studied with gram-positive Staphylococcus aureus (Sa) and gram-negative Escherichia coli (E. coli) bacteria. Sa was used as the test bacterium, and the antibacterial ability of NSPTICU implanted in rats was studied by gross view specimen collection, bacterial colony counting, HE staining and Giemsa staining. Results: Alizarin red staining, alkaline phosphatase (ALP) staining, quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis showed that NSPTICU promoted the osteogenic differentiation of MC3T3-E1 cells. The in vitro antimicrobial results showed that the NSPTICU implants exhibited better antibacterial properties. Animal experiments showed that NSPTICU can inhibit inflammation and promote the repair of bone defects. Conclusion: NSPTICU has excellent antibacterial and bone induction ability, and has broad application prospects in the treatment of bone defects related to orthopedics and dental sciences.

The Copper Efflux Regulator (CueR).

The copper efflux regulator (CueR) is a classical member of the MerR family of metalloregulators and is common in gram-negative bacteria. Through its C-terminal effector-binding domain, CueR senses cytoplasmic copper ions to regulate the transcription of genes contributing to copper homeostasis, an essential process for survival of all cells. In this chapter, we review the regulatory roles of CueR in the model organism Escherichia coli and the mechanisms for CueR in copper binding, DNA recognition, and interplay with RNA polymerase in regulating transcription. In light of biochemical and structural analyses, we provide molecular details for how CueR represses transcription in the absence of copper ions, how copper ions mediate CueR conformational change to form holo CueR, and how CueR bends and twists promoter DNA to activate transcription. We also characterize the functional domains and key residues involved in these processes. Since CueR is a representative member of the MerR family, elucidating its regulatory mechanisms could help to understand the CueR-like regulators in other organisms and facilitate the understanding of other metalloregulators in the same family.

Building the Bacterial Divisome at the Septum.

Across living organisms, division is necessary for cell survival and passing heritable information to the next generation. For this reason, cell division is highly conserved among eukaryotes and prokaryotes. Among the most highly conserved cell division proteins in eukaryotes are tubulin and actin. Tubulin polymerizes to form microtubules, which assemble into cytoskeletal structures in eukaryotes, such as the mitotic spindle that pulls chromatids apart during mitosis. Actin polymerizes to form a morphological framework for the eukaryotic cell, or cytoskeleton, that undergoes reorganization during mitosis. In prokaryotes, two of the most highly conserved cell division proteins are the tubulin homolog FtsZ and the actin homolog FtsA. In this chapter, the functions of the essential bacterial cell division proteins FtsZ and FtsA and their roles in assembly of the divisome at the septum, the site of cell division, will be discussed. In most bacteria, including Escherichia coli, the tubulin homolog FtsZ polymerizes at midcell, and this step is crucial for recruitment of many other proteins to the division site. For this reason, both FtsZ abundance and polymerization are tightly regulated by a variety of proteins. The actin-like FtsA protein polymerizes and tethers FtsZ polymers to the cytoplasmic membrane. Additionally, FtsA interacts with later stage cell division proteins, which are essential for division and for building the new cell wall at the septum. Recent studies have investigated how actin-like polymerization of FtsA on the lipid membrane may impact division, and we will discuss this and other ways that division in bacteria is regulated through FtsZ and FtsA.

Scarless Baculovirus Genome Editing Using Lambda-Red Recombineering in E. coli.

Baculoviruses are widely used for their potential as biological pesticide and as platform for the production of recombinant proteins and gene therapy vectors. The Baculovirus Expression Vector System (BEVS) is used for high level of expression of (multiple) proteins in insect cells. Baculovirus recombinants can be quickly constructed by transposition of the gene(s) of interest into a so-called bacmid, which is a baculovirus infectious clone maintained as single-copy, bacterial artificial chromosome in Escherichia coli. A two-step homologous recombineering technique using the lambda-red system in E. coli allows for scarless editing of the bacmid with PCR products based on sequence homology. In the first step, a selection cassette with 50 bp homology arms, typically generated by PCR, is inserted into the designated locus. In the second step, the selection cassette is removed based on a negative selection marker, such as SacB or rpsL. This lambda-red recombineering technique can be used for multiple gene editing purposes, including (large) deletions, insertions, and even single point mutations. Moreover, since there are no remnants of the editing process, successive modifications of the same bacmid are possible. This chapter provides detailed instructions to design and perform two-step homologous recombineering of baculovirus bacmid DNA in E. coli. We present two case studies demonstrating the utility of this technique for creating a deletion mutant of the chitinase and cathepsin genes and for introducing a single point mutation in the baculovirus gene gp41. This scarless genome editing approach can facilitate functional studies of baculovirus genes and improve the production of recombinant proteins using the BEVS.