Genes encoding a novel thermostable bacteriocin in the thermophilic bacterium Aeribacillus pallidus PI8.

AIM: Aeribacillus pallidus PI8 is a Gram-positive thermophilic bacterium that produces thermostable antimicrobial substances against several bacterial species, including Geobacillus kaustophilus HTA426. In the present study, we sought to identify genes of PI8 with antibacterial activity. METHODS AND RESULTS: We isolated, cloned, and characterized a thermostable bacteriocin from A. pallidus PI8 and named it pallidocyclin. Mass spectrometric analyses of pallidocyclin revealed that it had a circular peptide structure, and its precursor was encoded by pcynA in the PI8 genome. pcynA is the second gene within the pcynBACDEF operon. Expression of the full-length pcynBACDEF operon in Bacillus subtilis produced intact pallidocyclin, whereas expression of pcynF in G. kaustophilus HTA426 conferred resistance to pallidocyclin. CONCLUSION: Aeribacillus pallidus PI8 possesses the pcynBACDEF operon to produce pallidocyclin. pcynA encodes the pallidocyclin precursor, and pcynF acts as an antagonist of pallidocyclin.

Bacterial and fungal bioburden reduction on material surfaces using various sterilization techniques suitable for spacecraft decontamination.

Planetary protection is a guiding principle aiming to prevent microbial contamination of the solar system by spacecraft (forward contamination) and extraterrestrial contamination of the Earth (backward contamination). Bioburden reduction on spacecraft, including cruise and landing systems, is required to prevent microbial contamination from Earth during space exploration missions. Several sterilization methods are available; however, selecting appropriate methods is essential to eliminate a broad spectrum of microorganisms without damaging spacecraft components during manufacturing and assembly. Here, we compared the effects of different bioburden reduction techniques, including dry heat, UV light, isopropyl alcohol (IPA), hydrogen peroxide (H2O2), vaporized hydrogen peroxide (VHP), and oxygen and argon plasma on microorganisms with different resistance capacities. These microorganisms included Bacillus atrophaeus spores and Aspergillus niger spores, Deinococcus radiodurans, and Brevundimonas diminuta, all important microorganisms for considering planetary protection. Bacillus atrophaeus spores showed the highest resistance to dry heat but could be reliably sterilized (i.e., under detection limit) through extended time or increased temperature. Aspergillus niger spores and D. radiodurans were highly resistant to UV light. Seventy percent of IPA and 7.5% of H2O2 treatments effectively sterilized D. radiodurans and B. diminuta but showed no immediate bactericidal effect against B. atrophaeus spores. IPA immediately sterilized A. niger spores, but H2O2 did not. During VHP treatment under reduced pressure, viable B. atrophaeus spores and A. niger spores were quickly reduced by approximately two log orders. Oxygen plasma sterilized D. radiodurans but did not eliminate B. atrophaeus spores. In contrast, argon plasma sterilized B. atrophaeus but not D. radiodurans. Therefore, dry heat could be used for heat-resistant component bioburden reduction, and VHP or plasma for non-heat-resistant components in bulk bioburden reduction. Furthermore, IPA, H2O2, or UV could be used for additional surface bioburden reduction during assembly and testing. The systemic comparison of sterilization efficiencies under identical experimental conditions in this study provides basic criteria for determining which sterilization techniques should be selected during bioburden reduction for forward planetary protection.

A critical role of the periplasm in copper homeostasis in Gram-negative bacteria.

Copper is essential for life, but is toxic in excess. Copper homeostasis is achieved in the cytoplasm and the periplasm as a unique feature of Gram-negative bacteria. Especially, it has become clear the role of the periplasm and periplasmic proteins regarding whole-cell copper homeostasis. Here, we addressed the role of the periplasm and periplasmic proteins in copper homeostasis using a Systems Biology approach integrating experiments with models. Our analysis shows that most of the copper-bound molecules localize in the periplasm but not cytoplasm, suggesting that Escherichia coli utilizes the periplasm to sense the copper concentration in the medium and sequester copper ions. In particular, a periplasmic multi-copper oxidase CueO and copper-responsive transcriptional factor CusS contribute both to protection against Cu(I) toxicity and to incorporating copper into the periplasmic components/proteins. We propose that Gram-negative bacteria have evolved mechanisms to sense and store copper in the periplasm to expand their living niches.

Transcranial electrical stimulation technique for induction of unilateral motor evoked potentials.

OBJECTIVE: Transcranial electrical stimulation motor evoked potentials (TES-MEP) are widely used to monitor motor function; however, broad current spread and induced body movement are limitations of this technique. We herein report a localized stimulation technique for TES-MEP that induces unilateral MEP responses. METHODS: The stimulation of C1(+)-C4(-) or C2(+)-C3(-) was performed to induce right- or left-sided muscle contraction, respectively, in 70 patients. Electromyography was recorded by placing electrodes on the bilateral abductor pollicis brevis (APB) and abductor hallucis (AH) muscles. Stimulation conditions were regulated in the range to induce unilateral muscle contractions contralateral to the anodal stimulation. The thresholds and amplitudes of TES-MEP were retrospectively analyzed. RESULTS: The thresholds of APB were lower than those of AH in 47 patients, AH thresholds were lower than those of APB in 6 patients, and both APB and AH started to respond at the same intensity in 15 patients. This technical stimulation induced contralateral limb contractions with a suprathreshold stimulation of 129.4 ± 35.6 mA (mean ± standard deviation) in 68 patients (97%). Amplitudes in the suprathreshold stimulation of APB and AH responses were 727.5 ± 695.7 and 403.3 ± 325.7 µV, respectively. CONCLUSIONS: The C1(+)-C4/C2(+)-C3(-) stimulation in TES-MEP enables a localized stimulation to induce unilateral MEP responses. SIGNIFICANCE: Our stimulation technique enables the stable and safe monitoring of unilateral limbs, and contributes to the reliable monitoring of motor function in neurosurgery.

Bacillus velezensis S141, a soybean growth-promoting bacterium, hydrolyzes isoflavone glycosides into aglycones.

Bacillus velezensis S141, a plant growth-promoting rhizobacteria (PGPR), was isolated from a soybean field in Thailand. Previous studies demonstrated that S141 enhanced soybean growth, stimulating nodulation for symbiotic nitrogen fixation with soybean root nodule bacteria, including Bradyrhizobium diazoefficience USDA110. Isoflavone glycosides are produced in soybean roots and hydrolyzed into their aglycones, triggering nodulation. This study revealed that S141 efficiently hydrolyzed two isoflavone glycosides in soybean roots (daidzin and genistin) to their aglycones (daidzein and genistein, respectively). However, S141, Bacillus subtilis 168, NCIB3610, and B. velezensis FZB42 hydrolyzed isoflavone glucosides into aglycones. A BLASTp search suggested that S141 and the other three strains shared four genes encoding ß-glucosidases corresponding to bglA, bglC, bglH, and gmuD in B. subtilis 168. The gene inactivation analysis of B. subtilis 168 revealed that bglC encoded the major ß-glucosidase, contributing about half of the total activity to hydrolyze isoflavone glycosides and that bglA, bglH, and gmuD, all barely committed to the hydrolysis of isoflavone glycosides. Thus, an unknown ß-glucosidase exists, and our genetic knowledge of ß-glucosidases was insufficient to evaluate the ability to hydrolyze isoflavone glycosides. Nevertheless, S141 could predominate in the soybean rhizosphere, releasing isoflavone aglycones to enhance soybean nodulation.

Constitutive glucose dehydrogenase elevates intracellular NADPH levels and luciferase luminescence in Bacillus subtilis.

BACKGROUND: Genetic modifications in Bacillus subtilis have allowed the conversion of myo-inositol into scyllo-inositol, which is proposed as a therapeutic agent for Alzheimer's disease. This conversion comprises two reactions catalyzed by two distinct inositol dehydrogenases, IolG and IolW. The IolW-mediated reaction requires the intracellular regeneration of NADPH, and there appears to be a limit to the endogenous supply of NADPH, which may be one of the rate-determining factors for the conversion of inositol. The primary mechanism of NADPH regeneration in this bacterium remains unclear. RESULTS: The gdh gene of B. subtilis encodes a sporulation-specific glucose dehydrogenase that can use NADP+ as a cofactor. When gdh was modified to be constitutively expressed, the intracellular NADPH level was elevated, increasing the conversion of inositol. In addition, the bacterial luciferase derived from Photorhabdus luminescens became more luminescent in cells in liquid culture and colonies on culture plates. CONCLUSION: The results indicated that the luminescence of luciferase was representative of intracellular NADPH levels. Luciferase can therefore be employed to screen for mutations in genes involved in NADPH regeneration in B. subtilis, and artificial manipulation to enhance NADPH regeneration can promote the production of substances such as scyllo-inositol.

A New Tool for the Flexible Genetic Manipulation of Geobacillus kaustophilus.

Geobacillus kaustophilus , a thermophilic Gram-positive bacterium, is an attractive host for the development of high-temperature bioprocesses. However, its reluctance against genetic manipulation by standard methodologies hampers its exploitation. Here, we describe a simple methodology in which an artificial DNA segment on the chromosome of Bacillus subtilis can be transferred via pLS20-mediated conjugation resulting in subsequent integration in the genome of G. kaustophilus. Therefore, we have developed a transformation strategy to design an artificial DNA segment on the chromosome of B. subtilis and introduce it into G. kaustophilus . The artificial DNA segment can be freely designed by taking advantage of the plasticity of the B. subtilis genome and combined with the simplicity of pLS20 conjugation transfer. This transformation strategy would adapt to various Gram-positive bacteria other than G. kaustophilus . Graphical abstract.

Constitutive expression of the global regulator AbrB restores the growth defect of a genome-reduced Bacillus subtilis strain and improves its metabolite production.

Partial bacterial genome reduction by genome engineering can improve the productivity of various metabolites, possibly via deletion of non-essential genome regions involved in undesirable metabolic pathways competing with pathways for the desired end products. However, such reduction may cause growth defects. Genome reduction of Bacillus subtilis MGB874 increases the productivity of cellulases and proteases but reduces their growth rate. Here, we show that this growth defect could be restored by silencing redundant or less important genes affecting exponential growth by manipulating the global transcription factor AbrB. Comparative transcriptome analysis revealed that AbrB-regulated genes were upregulated and those involved in central metabolic pathway and synthetic pathways of amino acids and purine/pyrimidine nucleotides were downregulated in MGB874 compared with the wild-type strain, which we speculated were the cause of the growth defects. By constitutively expressing high levels of AbrB, AbrB regulon genes were repressed, while glycolytic flux increased, thereby restoring the growth rate to wild-type levels. This manipulation also enhanced the productivity of metabolites including γ-polyglutamic acid. This study provides the first evidence that undesired features induced by genome reduction can be relieved, at least partly, by manipulating a global transcription regulation system. A similar strategy could be applied to other genome engineering-based challenges aiming toward efficient material production in bacteria.

Functional analysis of a gene cluster for putative bacteriocin deduced from the genome sequence of Aeribacillus pallidus PI8.

Bacteriocins are a large family of peptides synthesized ribosomally by a variety of bacterial species. The genome of one of the thermophilic Gram-positive bacteria, Aeribacillus pallidus PI8, was found to possess an operon comprising five genes possibly involved in the production of a putative bacteriocin that was named pcnABCDE for the production of "pallidocyclicin." This study investigated the function of the pcn operon experimentally. The heterologous expression of the entire pcn operon from the plasmid was toxic to Escherichia coli but not to Bacillus subtilis. However, when the entire pcn operon was expressed constitutively, even the growth of B. subtilis was impaired, and at least pcnA was implied to serve as the precursor of pallidocyclicin. In addition, a strain of B. subtilis expressing the entire pcn operon from the plasmid showed toxicity to another thermophilic species, Geobacillus kaustophilus, at elevated temperatures, whereas another strain lacking pcnE alone from the pcn operon lost the toxicity, suggesting that pcnE might be involved in the biosynthesis of pallidocyclicin when it is produced in B. subtilis.

A novel method for transforming Geobacillus kaustophilus with a chromosomal segment of Bacillus subtilis transferred via pLS20-dependent conjugation.

BACKGROUND: Geobacillus kaustophilus is a thermophilic Gram-positive bacterium. Methods for its transformation are still under development. Earlier studies have demonstrated that pLS20catΔoriT mobilized the resident mobile plasmids from Bacillus subtilis to G. kaustophilus and transferred long segments of chromosome from one cell to another between B. subtilis. RESULTS: In this study, we applied mobilization of the B. subtilis chromosome mediated by pLS20catΔoriT to transform G. kaustophilus. We constructed a gene cassette to be integrated into G. kaustophilus and designed it within the B. subtilis chromosome. The pLS20catΔoriT-mediated conjugation successfully transferred the gene cassette from the B. subtilis chromosome into the G. kaustophilus allowing for the desired genetic transformation. CONCLUSIONS: This transformation approach described here will provide a new tool to facilitate the flexible genetic manipulation of G. kaustophilus.

Assessment of Bacillus subtilis Plasmid pLS20 Conjugation in the Absence of Quorum Sensing Repression.

Bacillus subtilis conjugative plasmid pLS20 uses a quorum-sensing mechanism to control expression levels of its conjugation genes, involving the repressor RcopLS20, the anti-repressor RappLS20, and the signaling peptide Phr*pLS20. In previous studies, artificial overexpression of rappLS20 in the donor cells was shown to enhance conjugation efficiency. However, we found that the overexpression of rappLS20 led to various phenotypic traits, including cell aggregation and death, which might have affected the correct determination of the conjugation efficiency when determined by colony formation assay. In the current study, conjugation efficiencies were determined under different conditions using a two-color fluorescence-activated flow cytometry method and measuring a single-round of pLS20-mediated transfer of a mobilizable plasmid. Under standard conditions, the conjugation efficiency obtained by fluorescence-activated flow cytometry was 23-fold higher than that obtained by colony formation. Furthermore, the efficiency difference increased to 45-fold when rappLS20 was overexpressed.

Identification of genes encoding a novel ABC transporter in Lactobacillus delbrueckii for inulin polymers uptake.

Lactobacillus delbrueckii JCM 1002T grows on highly polymerized inulin-type fructans as its sole carbon source. When it was grown on inulin, a > 10 kb long gene cluster inuABCDEF (Ldb1381-1386) encoding a plausible ABC transporter was suggested to be induced, since a transcriptome analysis revealed that the fourth gene inuD (Ldb1384) was up-regulated most prominently. Although Bacillus subtilis 168 is originally unable to utilize inulin, it became to grow on inulin upon heterologous expression of inuABCDEF. When freshly cultured cells of the recombinant B. subtilis were then densely suspended in buffer containing inulin polymers and incubated, inulin gradually disappeared from the buffer and accumulated in the cells without being degraded, whereas levan-type fructans did not disappear. The results imply that inuABCDEF might encode a novel ABC transporter in L. delbrueckii to "monopolize" inulin polymers selectively, thereby, providing a possible advantage in competition with other concomitant inulin-utilizing bacteria.

Genome sequences of two strains of Lactococcus lactis subsp. cremoris with the same ancestry but a different capacity to produce exopolysaccharides.

Strains of Lactococcus lactis subsp. cremoris are used to produce yogurt containing exopolysaccharides with a sticky texture. When strain G3-2 producing exopolysaccharides was grown at elevated temperatures, a spontaneous mutant EPSC, which had lost exopolysaccharides biosynthesis, was isolated. Genomes of the two strains were determined to be composed of a 2.4-Mb chromosome and up to eleven plasmids, and it was revealed that one of the plasmids encoding the gene cluster for exopolysaccharides biosynthesis was lost selectively in EPSC.

Identification of a repressor for the two iol operons required for inositol catabolism in Geobacillus kaustophilus.

Geobacillus kaustophilus HTA426, a thermophilic Gram-positive bacterium, feeds on inositol as its sole carbon source, and an iol gene cluster required for inositol catabolism has been postulated with reference to the iol genes in Bacillus subtilis. The iol gene cluster of G. kaustophilus comprises two tandem operons induced in the presence of inositol; however, the mechanism underlying this induction remains unclear. B. subtilis iolQ is known to be involved in the regulation of iolX encoding scyllo-inositol dehydrogenase, and its homologue in HTA426 was found two genes upstream of the first gene (gk1899) of the iol gene cluster and was termed iolQ in G. kaustophilus. When iolQ was inactivated in G. kaustophilus, not only cellular myo-inositol dehydrogenase activity due to gk1899 expression but also the transcription of the two iol operons became constitutive. IolQ was produced and purified as a C-terminal histidine (His)-tagged fusion protein in Escherichia coli and subjected to an in vitro gel electrophoresis mobility shift assay to examine its DNA-binding property. It was observed that IolQ bound to the DNA fragments containing each of the two iol promoter regions and that DNA binding was antagonized by myo-inositol. Moreover, DNase I footprinting analyses identified two tandem binding sites of IolQ within each of the iol promoter regions. By comparing the sequences of the binding sites, a consensus sequence for IolQ binding was deduced to form a palindrome of 5'-RGWAAGCGCTTSCY-3' (where R=A or G, W=A or T, S=G or C, and Y=C or T). IolQ functions as a transcriptional repressor regulating the induction of the two iol operons responding to myo-inositol.

Complete Genome Sequence of Nitrogen-Fixing Paenibacillus sp. Strain URB8-2, Isolated from the Rhizosphere of Wild Grass.

We report here the complete genome sequence of nitrogen-fixing Paenibacillus sp. strain URB8-2, isolated from the rhizosphere of wild grass in Kobe, Japan, revealing that this bacterium is related to Paenibacillus rhizophilus 7197, a novel species collected recently in Inner Mongolia, China, and that it possesses two gene clusters for distinct types of nitrogenases.

Complete Genome Sequence of Thermophilic Bacterium Aeribacillus pallidus PI8.

Here, we report the complete genome sequence of Aeribacillus pallidus PI8, a thermophilic bacterium, isolated from soybean stem extract. The sequence was determined using Illumina and Nanopore sequencers. Bioinformatic analyses of the genome sequence revealed the presence of possible bacteriocin gene clusters.

A bacterial cell factory converting glucose into scyllo-inositol, a therapeutic agent for Alzheimer's disease.

A rare stereoisomer of inositol, scyllo-inositol, is a therapeutic agent that has shown potential efficacy in preventing Alzheimer's disease. Mycobacterium tuberculosis ino1 encoding myo-inositol-1-phosphate (MI1P) synthase (MI1PS) was introduced into Bacillus subtilis to convert glucose-6-phosphate (G6P) into MI1P. We found that inactivation of pbuE elevated intracellular concentrations of NAD+·NADH as an essential cofactor of MI1PS and was required to activate MI1PS. MI1P thus produced was dephosphorylated into myo-inositol by an intrinsic inositol monophosphatase, YktC, which was subsequently isomerized into scyllo-inositol via a previously established artificial pathway involving two inositol dehydrogenases, IolG and IolW. In addition, both glcP and glcK were overexpressed to feed more G6P and accelerate scyllo-inositol production. Consequently, a B. subtilis cell factory was demonstrated to produce 2 g L-1 scyllo-inositol from 20 g L-1 glucose. This cell factory provides an inexpensive way to produce scyllo-inositol, which will help us to challenge the growing problem of Alzheimer's disease in our aging society.

Production of scyllo-Inositol: Conversion of Rice Bran into a Promising Disease-Modifying Therapeutic Agent for Alzheimer's Disease.

scyllo-Inositol (SI) is one of the inositol stereoisomers, rare in the nature, and expected as a promising disease-modifying therapeutic agent for Alzheimer's disease. On the other hand, myo-inositol (MI) is another inositol stereoisomer most abundant in nature and thus supplied from agricultural byproducts including rice bran. Bacillus subtilis was genetically modified in its inositol metabolism and phytase secretion, to develope the bioconversion processes to produce SI from rice bran. Phytase, an enzyme that degrades phytate in rice bran into MI, was secreted in a B. subtilis strain with the optimized signal peptide. Another B. subtilis strain was constructed with the constitutive and simultaneous overexpression of IolG and IolW, which are the two inositol dehydrogenases responsible for the conversion, to demonstrate an efficient conversion of MI into SI with a rate up to 10 g/L/48 h. In order to devise further elevation in the conversion efficiency, we attempted to improve the substrate uptake by overexpressing iolT for the major MI transporter. In addition, Escherichia coli pntAB encoding the membrane-bound transhydrogenase was introduced aiming at enhanced supply of NADPH required for the rate-limiting IolW reaction. These additional modifications successfully elevated the conversion efficiency with an improved rate up to almost 30 g/L/48 h. Together with the improved phytase secretion, technological infrastructure for social implementation of SI production from rice bran is on the way.

Construction of Its Evaluation System in Originally Designed Test-Chamber System and Sporicidal Activity of Aerosolized Hypochlorite Solution to Bacillus subtilis Spores.

　Effective spatial disinfection systems are required for human health care, public hygiene, and food and medicine manufacturing. Although some aerosolized disinfectants were already applied to its purpose, accurate evaluation systems were under constructed. In this study, the spatial sporicidal activity of aerosolized hypochlorite solution (AHS) to dormant cells, Bacillus subtilis spores, was evaluated by an originally designed chamber system. In the test-chamber, AHS was supplied and existed as micro-droplets, and environmental relative humidity (RH) could be controlled. Available chlorine (AC) exposure was also controlled with appropriate AC loading but was influenced by the acidity of AHS. Our results indicated that inactivation of spore was depend on AC exposure amount and time. On the other hand, unsaturated environmental RH markedly decreased spore inactivation. This study indicated that our test-chamber system can provide reproducible test data under a homogeneous air condition, and, thereby, that the data obtained by the chamber system should contribute to predicting the AC-required dose to disinfect a whole building.

Bradyrhizobium diazoefficiens USDA110 PhaR functions for pleiotropic regulation of cellular processes besides PHB accumulation.

BACKGROUND: Bradyrhizobium diazoefficiens USDA110 nodulates soybeans for nitrogen fixation. It accumulates poly-3-hydroxybutyrate (PHB), which is of physiological importance as a carbon/energy source for survival during starvation, infection, and nitrogen fixation conditions. PHB accumulation is orchestrated by not only the enzymes for PHB synthesis but also PHB-binding phasin proteins (PhaPs) stabilizing the PHB granules. The transcription factor PhaR controls the phaP genes. RESULTS: Inactivation of phaR led to decreases in PHB accumulation, less cell yield, increases in exopolysaccharide (EPS) production, some improvement in heat stress tolerance, and slightly better growth under microaerobic conditions. Changes in the transcriptome upon phaR inactivation were analyzed. PhaR appeared to be involved in the repression of various target genes, including some PHB-degrading enzymes and others involved in EPS production. Furthermore, in vitro gel shift analysis demonstrated that PhaR bound to the promoter regions of representative targets. For the phaP1 and phaP4 promoter regions, PhaR-binding sites were determined by DNase I footprinting, allowing us to deduce a consensus sequence for PhaR-binding as TGCRNYGCASMA (R: A or G, Y: C or T, S: C or G, M: A or C). We searched for additional genes associated with a PhaR-binding sequence and found that some genes involved in central carbon metabolism, such as pdhA for pyruvate dehydrogenase and pckA for phosphoenolpyruvate carboxykinase, may be regulated positively and directly by PhaR. CONCLUSIONS: These results suggest that PhaR could regulate various genes not only negatively but also positively to coordinate metabolism holistically in response to PHB accumulation.