Beta-lactam antibiotics induce a lethal malfunctioning of the bacterial cell wall synthesis machinery.

Penicillin and related beta-lactams comprise one of our oldest and most widely used antibiotic therapies. These drugs have long been known to target enzymes called penicillin-binding proteins (PBPs) that build the bacterial cell wall. Investigating the downstream consequences of target inhibition and how they contribute to the lethal action of these important drugs, we demonstrate that beta-lactams do more than just inhibit the PBPs as is commonly believed. Rather, they induce a toxic malfunctioning of their target biosynthetic machinery involving a futile cycle of cell wall synthesis and degradation, thereby depleting cellular resources and bolstering their killing activity. Characterization of this mode of action additionally revealed a quality control function for enzymes that cleave bonds in the cell wall matrix. The results thus provide insight into the mechanism of cell wall assembly and suggest how best to interfere with the process for future antibiotic development.

Structural basis of flagellar motility regulation by the MogR repressor and the GmaR antirepressor in Listeria monocytogenes.

The pathogenic Listeria monocytogenes bacterium produces the flagellum as a locomotive organelle at or below 30°C outside the host, but it halts flagellar expression at 37°C inside the human host to evade the flagellum-induced immune response. Listeria monocytogenes GmaR is a thermosensor protein that coordinates flagellar expression by binding the master transcriptional repressor of flagellar genes (MogR) in a temperature-responsive manner. To understand the regulatory mechanism whereby GmaR exerts the antirepression activity on flagellar expression, we performed structural and mutational analyses of the GmaR-MogR system. At or below 30°C, GmaR exists as a functional monomer and forms a circularly enclosed multidomain structure via an interdomain interaction. GmaR in this conformation recognizes MogR using the C-terminal antirepressor domain in a unique dual binding mode and mediates the antirepressor function through direct competition and spatial restraint mechanisms. Surprisingly, at 37°C, GmaR rapidly forms autologous aggregates that are deficient in MogR neutralization capabilities.

One-step formation of three-dimensional interconnected T-shaped microstructures inside composites by orthogonal bidirectional self-assembly method.

The fillers inside a polymer matrix should typically be self-assembled in both the horizontal and vertical directions to obtain 3-dimentional (3D) percolation pathways, whereby the fields of application can be expanded and the properties of organic-inorganic composite films improved. Conventional dielectrophoresis techniques can typically only drive fillers to self-assemble in only one direction. We have devised a one-step dielectrophoresis-driven approach that effectively induces fillers self-assembly along two orthogonal axes, which results in the formation of 3D interconnected T-shaped iron microstructures (3D-T CIP) inside a polymer matrix. This approach to carbonyl iron powder (CIP) embedded in a polymer matrix results in a linear structure along the thickness direction and a network structure on the top surface of the film. The fillers in the polymer were controlled to achieve orthogonal bidirectional self-assembly using an external alternating current (AC) electric field and a non-contact technique that did not lead to electrical breakdown. The process of 3D-T CIP formation was observed in real time using in situ observation methods with optical microscopy, and the quantity and quality of self-assembly were characterized using statistical and fractal analysis. The process of fillers self-assembly along the direction perpendicular to the electric field was explained by finite element analogue simulations, and the results indicated that the insulating polyethylene terephthalate (PET) film between the electrode and the CIP/prepolymer suspension was the key to the formation of the 3D-T CIP. In contrast to the traditional two-step method of fabricating sandwich-structured film, the fabricated 3D-T CIP film with 3D electrically conductive pathways can be applied as magnetic field sensor.

A one-step electric field-induced self-assembly method was developed to efficiently control the self-assembly of fillers along two orthogonal axes to form three-dimensional interconnected T-shaped microstructure assembles of carbonyl iron powder inside a polymer matrix.

Enhanced capacitive pressure sensing performance by charge generation from filler movement in thin and flexible PVDF-GNP composite films.

This study introduces an approach to overcome the limitations of conventional pressure sensors by developing a thin and lightweight composite film specifically tailored for flexible capacitive pressure sensors, with a particular emphasis on the medium and high pressure range. To accomplish this, we have engineered a composite film by combining polyvinylidene fluoride (PVDF) and graphite nanoplatelets (GNP) derived from expanded graphite (Ex-G). A uniform sized GNPs with an average lateral size of 2.55av and an average thickness of 33.74 av with narrow size distribution was obtained with a gas-induced expansion of expandable graphite (EXP-G) combined with tip sonication in solvent. By this precisely controlled GNP within the composite film, a remarkable improvement in sensor sensitivity has been achieved, surpassing 4.18 MPa-1 within the pressure range of 0.1 to 1.6 MPa. This enhancement can be attributed to the generation of electric charge from the movement of GNP in the polymer matrix. Additionally, stability testing has demonstrated the reliable operation of the composite film over 1000 cycles. Notably, the composite film exhibits exceptional continuous pressure sensing capabilities with a rapid response time of approximately 100 milliseconds. Experimental validation using a 3 × 3 sensor array has confirmed the accurate detection of specific contact points, thus highlighting the potential of the composite film in selective pressure sensing. These findings signify an advancement in the field of flexible capacitive pressure sensors that offer enhanced sensitivity, consistent operation, rapid response time, and the unique ability to selectively sense pressure.

Structural analysis of the virulence gene protein IceA2 from Helicobacter pylori.

Helicobacter pylori is a pathogenic bacterium that causes gastric ulcers and cancer. Among the diverse virulence genes of H. pylori, the IceA gene was identified to be expressed upon adherence to host cells. The IceA gene has two alleles, iceA1 and iceA2, which encode completely different proteins. IceA1 protein was shown to exert endonuclease activity, whereas IceA2 has never been analyzed at the molecular level. Based on a sequence analysis, IceA2 proteins differ in length depending on the strain and are classified into five groups (A-E). To structurally characterize IceA2, we determined the crystal structure of group-D IceA2 (IceA2sD) and performed a modeling-based comparative analysis of IceA2 groups. IceA2sD consists of three ß-sheet repeats and serially arranges them like the ß-propeller structure of the WD40 domain. However, each ß-sheet of IceA2 is stabilized using a unique structural motif that is not observed in WD40. Moreover, IceA2sD lacks an additionally appended ß-strand and does not form the Velcro-like closure of WD40. Therefore, IceA2sD adopts a curved rod-like structure rather than an enclosed circular structure in WD40. IceA2 proteins contain 1-4 ß-sheet modules depending on the groups and are modeled to be highly diverse in size and shape.

A central role for PBP2 in the activation of peptidoglycan polymerization by the bacterial cell elongation machinery.

Cell elongation in rod-shaped bacteria is mediated by the Rod system, a conserved morphogenic complex that spatially controls cell wall assembly by the glycan polymerase RodA and crosslinking enzyme PBP2. Using Escherichia coli as a model system, we identified a PBP2 variant that promotes Rod system function when essential accessory components of the machinery are inactivated. This PBP2 variant hyperactivates cell wall synthesis in vivo and stimulates the activity of RodA-PBP2 complexes in vitro. Cells with the activated synthase also exhibited enhanced polymerization of the actin-like MreB component of the Rod system. Our results define an activation pathway governing Rod system function in which PBP2 conformation plays a central role in stimulating both glycan polymerization by its partner RodA and the formation of cytoskeletal filaments of MreB to orient cell wall assembly. In light of these results, previously isolated mutations that activate cytokinesis suggest that an analogous pathway may also control cell wall synthesis by the division machinery.

The mecillinam resistome reveals a role for peptidoglycan endopeptidases in stimulating cell wall synthesis in Escherichia coli.

Bacterial cells are typically surrounded by an net-like macromolecule called the cell wall constructed from the heteropolymer peptidoglycan (PG). Biogenesis of this matrix is the target of penicillin and related beta-lactams. These drugs inhibit the transpeptidase activity of PG synthases called penicillin-binding proteins (PBPs), preventing the crosslinking of nascent wall material into the existing network. The beta-lactam mecillinam specifically targets the PBP2 enzyme in the cell elongation machinery of Escherichia coli. Low-throughput selections for mecillinam resistance have historically been useful in defining mechanisms involved in cell wall biogenesis and the killing activity of beta-lactam antibiotics. Here, we used transposon-sequencing (Tn-Seq) as a high-throughput method to identify nearly all mecillinam resistance loci in the E. coli genome, providing a comprehensive resource for uncovering new mechanisms underlying PG assembly and drug resistance. Induction of the stringent response or the Rcs envelope stress response has been previously implicated in mecillinam resistance. We therefore also performed the Tn-Seq analysis in mutants defective for these responses in addition to wild-type cells. Thus, the utility of the dataset was greatly enhanced by determining the stress response dependence of each resistance locus in the resistome. Reasoning that stress response-independent resistance loci are those most likely to identify direct modulators of cell wall biogenesis, we focused our downstream analysis on this subset of the resistome. Characterization of one of these alleles led to the surprising discovery that the overproduction of endopeptidase enzymes that cleave crosslinks in the cell wall promotes mecillinam resistance by stimulating PG synthesis by a subset of PBPs. Our analysis of this activation mechanism suggests that, contrary to the prevailing view in the field, PG synthases and PG cleaving enzymes need not function in multi-enzyme complexes to expand the cell wall matrix.

Pathway-Directed Screen for Inhibitors of the Bacterial Cell Elongation Machinery.

New antibiotics are needed to combat the growing problem of resistant bacterial infections. An attractive avenue toward the discovery of such next-generation therapies is to identify novel inhibitors of clinically validated targets, like cell wall biogenesis. We have therefore developed a pathway-directed whole-cell screen for small molecules that block the activity of the Rod system of Escherichia coli This conserved multiprotein complex is required for cell elongation and the morphogenesis of rod-shaped bacteria. It is composed of cell wall synthases and membrane proteins of unknown function that are organized by filaments of the actin-like MreB protein. Our screen takes advantage of the conditional essentiality of the Rod system and the ability of the beta-lactam mecillinam (also known as amdinocillin) to cause a toxic malfunctioning of the machinery. Rod system inhibitors can therefore be identified as molecules that promote growth in the presence of mecillinam under conditions permissive for the growth of Rod- cells. A screen of ∼690,000 compounds identified 1,300 compounds that were active against E. coli Pathway-directed screening of a majority of this subset of compounds for Rod inhibitors successfully identified eight analogs of the MreB antagonist A22. Further characterization of the A22 analogs identified showed that their antibiotic activity under conditions where the Rod system is essential was strongly correlated with their ability to suppress mecillinam toxicity. This result combined with those from additional biological studies reinforce the notion that A22-like molecules are relatively specific for MreB and suggest that the lipoprotein transport factor LolA is unlikely to be a physiologically relevant target as previously proposed.

Identification of MltG as a potential terminase for peptidoglycan polymerization in bacteria.

Bacterial cells are fortified against osmotic lysis by a cell wall made of peptidoglycan (PG). Synthases called penicillin-binding proteins (PBPs), the targets of penicillin and related antibiotics, polymerize the glycan strands of PG and crosslink them into the cell wall meshwork via attached peptides. The average length of glycan chains inserted into the matrix by the PBPs is thought to play an important role in bacterial morphogenesis, but polymerization termination factors controlling this process have yet to be discovered. Here, we report the identification of Escherichia coli MltG (YceG) as a potential terminase for glycan polymerization that is broadly conserved in bacteria. A clone containing mltG was initially isolated in a screen for multicopy plasmids generating a lethal phenotype in cells defective for the PG synthase PBP1b. Biochemical studies revealed that MltG is an inner membrane enzyme with endolytic transglycosylase activity capable of cleaving at internal positions within a glycan polymer. Radiolabeling experiments further demonstrated MltG-dependent nascent PG processing in vivo, and bacterial two-hybrid analysis identified an MltG-PBP1b interaction. Mutants lacking MltG were also shown to have longer glycans in their PG relative to wild-type cells. Our combined results are thus consistent with a model in which MltG associates with PG synthetic complexes to cleave nascent polymers and terminate their elongation.

Room-Temperature H2 Gas Sensing Characterization of Graphene-Doped Porous Silicon via a Facile Solution Dropping Method.

In this study, a graphene-doped porous silicon (G-doped/p-Si) substrate for low ppm H2 gas detection by an inexpensive synthesis route was proposed as a potential noble graphene-based gas sensor material, and to understand the sensing mechanism. The G-doped/p-Si gas sensor was synthesized by a simple capillary force-assisted solution dropping method on p-Si substrates, whose porosity was generated through an electrochemical etching process. G-doped/p-Si was fabricated with various graphene concentrations and exploited as a H2 sensor that was operated at room temperature. The sensing mechanism of the sensor with/without graphene decoration on p-Si was proposed to elucidate the synergetic gas sensing effect that is generated from the interface between the graphene and p-type silicon.

Identification of the SlmA active site responsible for blocking bacterial cytokinetic ring assembly over the chromosome.

Bacterial cells use chromosome-associated division inhibitors to help coordinate the processes of DNA replication and segregation with cytokinesis. SlmA from Escherichia coli, a member of the tetracycline repressor (TetR)-like protein family, is one example of this class of regulator. It blocks the assembly of the bacterial cytokinetic ring by interfering with the polymerization of the tubulin-like FtsZ protein in a manner that is dramatically stimulated upon specific DNA binding. Here we used a combination of molecular genetics and biochemistry to identify the active site of SlmA responsible for disrupting FtsZ polymerization. Interestingly, this site maps to a region of SlmA that in the published DNA-free structure is partially occluded by the DNA-binding domains. In this conformation, the SlmA structure resembles the drug/inducer-bound conformers of other TetR-like proteins, which in the absence of inducer require an inward rotation of their DNA-binding domains to bind successive major grooves on operator DNA. Our results are therefore consistent with a model in which DNA-binding activates SlmA by promoting a rotational movement of the DNA-binding domains that fully exposes the FtsZ-binding sites. SlmA may thus represent a special subclass of TetR-like proteins that have adapted conformational changes normally associated with inducer sensing in order to modulate an interaction with a partner protein. In this case, the adaptation ensures that SlmA only blocks cytokinesis in regions of the cell occupied by the origin-proximal portion of the chromosome where SlmA-binding sites are enriched.

Effective gene delivery into adipose-derived stem cells: transfection of cells in suspension with the use of a nuclear localization signal peptide-conjugated polyethylenimine.

BACKGROUND AIMS: Adipose-derived stem cells have the ability to turn into several clinically important cell types. However, it is difficult to transfect these cells with the use of conventional cationic lipid-based reagents. Polyethylenimine (PEI) is considered to be an inexpensive and effective tool for delivery of nucleic acids into mammalian cells. METHODS: We used a linear PEI conjugated with the nuclear localization signal (NLS) peptide of Simian vacuolating virus 40 large T antigen (PEI-NLS) for transfection of plasmid DNA into adipose-derived cells. We also tested if transfection of cells in suspension might improve the degree and duration of exogenous gene expression. RESULTS: Transfection of cells in suspension with the use of a PEI conjugated with an NLS peptide resulted in high levels of reporter gene expression for an extended period of time in clonal 3T3-L1 preadipocytes and native human adipose-derived stem cells. The reporter gene expression increased for 3 days after the addition of the PEI-NLS peptide-DNA mixture in cell suspension and remained significant for at least 7 days. Cell density did not influence the level of reporter gene expression. Thus, the suspension method with the use of an NLS peptide-conjugated PEI leads to a robust and sustained expression of exogenous genes in adipose-derived cells. CONCLUSIONS: The devised transfection method may be useful for reprogramming of adipose-derived stem cells and cell-based therapy.

Nucleoid occlusion factor SlmA is a DNA-activated FtsZ polymerization antagonist.

The tubulin-like FtsZ protein initiates assembly of the bacterial cytokinetic machinery by polymerizing into a ring structure, the Z ring, at the prospective site of division. To block Z-ring formation over the nucleoid and help coordinate cell division with chromosome segregation, Escherichia coli employs the nucleoid-associated division inhibitor, SlmA. Here, we investigate the mechanism by which SlmA regulates FtsZ assembly. We show that SlmA disassembles FtsZ polymers in vitro. In addition, using chromatin immunoprecipitation (ChIP), we identified 24 SlmA-binding sequences (SBSs) on the chromosome. Remarkably, SlmA binding to SBSs dramatically enhanced its ability to interfere with FtsZ polymerization, and ChIP studies indicate that SlmA regulates FtsZ assembly at these sites in vivo. Because of the dynamic and highly organized nature of the chromosome, coupling SlmA activation to specific DNA binding provides a mechanism for the precise spatiotemporal control of its anti-FtsZ activity within the cell.

Assembly of Bacterial Surface Glycopolymers as an Antibiotic Target.

Bacterial cells are covered with various glycopolymers such as peptidoglycan (PG), lipopolysaccharides (LPS), teichoic acids, and capsules. Among these glycopolymers, PG assembly is the target of some of our most effective antibiotics, consistent with its essentiality and uniqueness to bacterial cells. Biosynthesis of other surface glycopolymers have also been acknowledged as potential targets for developing therapies to control bacterial infections, because of their importance for bacterial survival in the host environment. Moreover, biosynthesis of most surface glycopolymers are closely related to PG assembly because the same lipid carrier is shared for glycopolymer syntheses. In this review, I provide an overview of PG assembly and antibiotics that target this pathway. Then, I discuss the implications of a common lipid carrier being used for assembly of PG and other surface glycopolymers in antibiotic development.

Treatment of Colistin Dependence-Developing Acinetobacter baumannii with Antibiotic Combinations at Subinhibitory Concentrations.

Recent studies have revealed that colistin dependence frequently develops in colistin-susceptible Acinetobacter baumannii isolates. Despite resistance in parental strains, colistin-dependent mutants showed increased susceptibility to several antibiotics, which suggests the possibility of developing strategies to eliminate multidrug-resistant (MDR) A. baumannii. We investigated in vitro and in vivo efficacy of combinations of colistin and other antibiotics using MDR A. baumannii strains H08-391, H06-855, and H09-94, which are colistin-susceptible but develops colistin dependence upon exposure to colistin. An in vitro time-killing assay, a checkerboard assay, and an antibiotic treatment assay using Galleria mellonella larvae were performed. Although a single treatment of colistin at a high concentration did not prevent colistin dependence, combinations of colistin with other antibiotics at subinhibitory concentrations, especially amikacin, eradicated the strains by inhibiting the development of colistin dependence, in the in vitro time-killing assay. Only 40% of G. mellonella larvae infected by A. baumannii survived with colistin treatment alone; however, all or most of them survived following treatment with the combination of colistin and other antibiotics (amikacin, ceftriaxone, and tetracycline). Our results suggest the possibility of the combination of colistin and amikacin or other antibiotics as one of therapeutic options against A. baumannii infections by eliminating colistin-dependent mutants.

The Tol-Pal System Plays an Important Role in Maintaining Cell Integrity During Elongation in Escherichia coli.

The Tol-Pal system is a transenvelope complex widely conserved among Gram-negative bacteria. It is recruited to the septal ring during cytokinesis, and its inactivation causes pleiotropic phenotypes mainly associated with the division process. From our genetic screen to identify factors required for delaying lysis upon treatment of beta lactams, we discovered that the tol-pal mutant shares similar defects with mutants of the major class A PBP system (PBP1b-LpoB) in terms of lysis prevention. Further phenotypic analyses revealed that the Tol-Pal system plays an important role in maintaining cell integrity not only during septation, but also during cell elongation. Simultaneous inactivation of the Tol-Pal system and the PBP1b-LpoB system leads to lysis during cell elongation as well as during division. Moreover, production of the Lpo activator-bypass PBP1b, but not wild-type PBP1b, partially suppressed the Tol-Pal defect in maintaining cell integrity upon treatment of mecillinam specific for the Rod system, suggesting that the Tol-Pal system is likely to be involved in the activation of aPBP by Lpo factors. Overall, our results indicate that the Tol-Pal system plays an important role in maintaining cell wall integrity during elongation in addition to its multifaceted roles during cytokinesis.

A Cell Wall Hydrolase MepH Is Negatively Regulated by Proteolysis Involving Prc and NlpI in Escherichia coli.

Cell wall assembly of Gram-negative bacteria requires DD-endopeptidase activity that cleaves peptidoglycan (PG) crosslinks in addition to PG synthetic activity, and the activity of DD-endopeptidases needs to be tightly regulated to maintain cell wall integrity during PG expansion. Among the major DD-endopeptidases functioning for PG assembly in Escherichia coli, MepS and MepM have been shown to be negatively controlled by the periplasmic protease Prc. In this study, we performed a genetic selection using the synthetic lethality between the mepS and mepM mutations in rich medium to uncover regulatory mechanisms controlling the activity of DD-endopeptidases other than MepS and MepM. This selection revealed mutations in prc and nlpI as suppressors. Gene deletion analyses revealed that MepH is required for suppression of the MepS- MepM- growth defect by the prc or nlpI mutation. We also discovered that MepH is directly degraded by Prc and that this degradation is further promoted by NlpI. Thus, our study showed that all three DD-endopeptidases which play major roles in PG assembly of E. coli under normal physiological conditions are controlled by a common periplasmic protease.

Comparative analysis of the Colistin resistance-regulating gene cluster in Klebsiella species.

CrrAB two-component regulatory system is associated with colistin resistance in Klebsiella pneumoniae. Recently, some K. pneumoniae isolates lacking crrAB genes have been identified. In this study, we investigated the distribution and structural variation of the crrBAC-kexD cluster. To evaluate the structural variation of the crrBAC-kexD cluster, we explored 59 clinical K. pneumoniae isolates from Korea, and 508 whole genomes of K. pneumoniae and other strains of Klebsiella sp. Significant structural variations in crrBAC-kexD and its surrounding regions were identified among K. pneumoniae genomes. Within the genus Klebsiella, the cluster was identified only in K. pneumoniae, K. variicola, and K. quasipneumoniae, which form the K. pneumoniae complex. Among the 304 available K. pneumoniae genomes, an intact crrBAC-kexD cluster was identified in 178 isolates (58.6%), while the cluster was absent in 90 isolates (29.6%). Partial deletions within the cluster were identified in 22 genomes (7.2%). The most diverse structural patterns of the crrBAC-kexD cluster were observed in ST11 strains. Some clades lacked the crrBAC-kexD cluster. The crrBAC-kexD cluster was identified in the genomes of other bacterial species, including Citrobacter freundii and Enterobacter ludwigii. The crrBAC-kexD cluster is proposed to have been acquired by the ancestor of the K. pneumoniae complex from other bacterial species and the cluster may have been lost and re-acquired repeatedly in K. pneumoniae strains according to the phylogenetic analysis. The dynamic evolution of the crrBAC-kexD cluster suggests that it may have other roles, in addition to colistin resistance, in bacterial physiology.

Overexpression of a DNA Methyltransferase Increases Persister Cell Formation in Acinetobacter baumannii.

Many mechanisms have been proposed to be involved in the formation of bacterial persister cells. In this study, we investigated the impact of dam encoding DNA methylation on persister cell formation in Acinetobacter. We constructed plasmids overexpressing dam encoding DNA-(adenine N6)-methyltransferase and four genes as possibly involved in persistence and introduced them into three A. baumannii strains. For persister cell formation assays, bacteria were exposed to ciprofloxacin, imipenem, cefotaxime, and rifampin, and the transcription levels of the genes were measured by qRT-PCR. In addition, growth curves of strains were determined. We found that all five genes were upregulated following antibiotic exposure. Dam overexpression increased persister cell formation rates and activated the four persister cell-involved genes. Among the four persister cell-involved genes, only RecC overexpression increase persister cell formation rates. While recC-overexpressing strains showed higher growth rates, dam-overexpressing strains showed decreased growth rates. In this study, we revealed that a DNA methyltransferase may regulate persister cell formation in A. baumannii, while RecC seems to mediate epigenetic regulation of persister cell formation. However, Dam and RecC may act at different persister cell formation states. IMPORTANCE Bacterial persister cells are not killed by high concentration of antibiotics, despite its antibiotic susceptibility. It has been known that they may cause antibiotic treatment failure and contribute to the evolution of antibiotic resistance. Although many mechanisms have been suggested and verified for persister cell formation, many remains to be uncovered. In this study, we report that DNA methyltransferase leads to an increase in persister cell formation, through transcriptional activation of several regulatory genes. Our results suggest that DNA methyltransferases could be target proteins to prevent formation of persister cells.

Microstructure Evolution in Plastic Deformed Bismuth Telluride for the Enhancement of Thermoelectric Properties.

Thermoelectric generators are solid-state energy-converting devices that are promising alternative energy sources. However, during the fabrication of these devices, many waste scraps that are not eco-friendly and with high material cost are produced. In this work, a simple powder processing technology is applied to prepare n-type Bi2Te3 pellets by cold pressing (high pressure at room temperature) and annealing the treatment with a canning package to recycle waste scraps. High-pressure cold pressing causes the plastic deformation of densely packed pellets. Then, the thermoelectric properties of pellets are improved through high-temperature annealing (500 ∘C) without phase separation. This enhancement occurs because tellurium cannot escape from the canning package. In addition, high-temperature annealing induces rapid grain growth and rearrangement, resulting in a porous structure. Electrical conductivity is increased by abnormal grain growth, whereas thermal conductivity is decreased by the porous structure with phonon scattering. Owing to the low thermal conductivity and satisfactory electrical conductivity, the highest ZT value (i.e., 1.0) is obtained by the samples annealed at 500 ∘C. Hence, the proposed method is suitable for a cost-effective and environmentally friendly way.