A search for acrolein scavengers among food components.

Brain stroke is a major cause of being bedridden for elderly people, and preventing stroke is important for maintaining quality of life (QOL). Acrolein is a highly reactive aldehyde and causes tissue damage during stroke. Decreasing acrolein toxicity ameliorates tissue injury during brain stroke. In this study, we tried to identify food components which decrease acrolein toxicity. We found that 2-furanmethanethiol, cysteine methyl and ethyl esters, alliin, lysine and taurine decreased acrolein toxicity. These compounds neutralized acrolein by direct interaction. However, the interaction between acrolein and taurine was not so strong. Approximately 30 mM taurine was necessary to interact with 10 µM acrolein, and 2 g/kg taurine was necessary to decrease the size of mouse brain infarction. Taurine also slightly increased polyamine contents, which are involved in decrease in the acrolein toxicity. Mitochondrial potential damage by acrolein was also protected by taurine. Our results indicate that daily intake of foods containing 2-furanmethanethiol, cysteine methyl and ethyl esters, alliin, lysine and taurine may prevent severe injury in brain stroke and improve the quality of life for elderly people.

Transient Receptor Potential Ankyrin 1 (TRPA1) Channel Mediates Acrolein Cytotoxicity in Human Lung Cancer Cells.

Transient receptor potential ankyrin 1 (TRPA1) is a nonselective ion channel implicated in thermosensation and inflammatory pain. It has been reported that expression of the TRPA1 channel is induced by cigarette smoke extract. Acrolein found in cigarette smoke is highly toxic and known as an agonist of the TRPA1 channel. However, the role of TRPA1 in the cytotoxicity of acrolein remains unclear. Here, we investigated whether the TRPA1 channel is involved in the cytotoxicity of acrolein in human lung cancer A549 cells. The IC50 of acrolein in A549 cells was 25 µM, and acrolein toxicity increased in a concentration- and time-dependent manner. When the effect of acrolein on TRPA1 expression was examined, the expression of TRPA1 in A549 cells was increased by treatment with 50 µM acrolein for 24 h or 500 µM acrolein for 30 min. AP-1, a transcription factor, was activated in the cells treated with 50 µM acrolein for 24 h, while induction of NF-κB and HIF-1α was observed in the cells treated with 500 µM acrolein for 30 min. These results suggest that acrolein induces TRPA1 expression by activating these transcription factors. Overexpression of TRPA1 in A549 cells increased acrolein sensitivity and the level of protein-conjugated acrolein (PC-Acro), while knockdown of TRPA1 in A549 cells or treatment with a TRPA1 antagonist caused tolerance to acrolein. These findings suggest that acrolein induces the TRPA1 channel and that an increase in TRPA1 expression promotes the cytotoxicity of acrolein.

Alkaline Stress Causes Changes in Polyamine Biosynthesis in Thermus thermophilus.

An extreme thermophile, Thermus thermophilus, produces 16 different polyamines including long-chain and branched-chain polyamines. The composition and content of polyamines in the thermophile cells change not only with growth temperature but also with pH changes. In particular, cell growth decreased greatly at alkaline medium together with significant changes in the composition and content of polyamines. The amounts of tetraamines (spermine and its homologs) markedly decreased at alkaline pH. Thus, we knocked out the speE gene, which is involved in the biosynthesis of tetraamines, and changes of composition of polyamines with pH changes in the mutant cells were studied. Cell growth in the ΔspeE strain was decreased compared with that of the wild-type strain for all pHs, suggesting that tetraamines are important for cell proliferation. Interestingly, the amount of spermidine decreased and that of putrescine increased in wild-type cells at elevated pH, although T. thermophilus lacks a putrescine synthesizing pathway. In addition, polyamines possessing a diaminobutane moiety, such as spermine, decreased greatly at high pH. We assessed whether the speB gene encoding aminopropylagmatine ureohydrolase (TtSpeB) is directly involved in the synthesis of putrescine. The catalytic assay of the purified enzyme indicated that TtSpeB accepts agmatine as its substrate and produces putrescine due to the change in substrate specificity at high pH. These results suggest that pH stress was exacerbated upon intracellular depletion of polyamines possessing a diaminobutane moiety induced by unusual changes in polyamine biosynthesis under high pH conditions.

Polyamines regulate gene expression by stimulating translation of histone acetyltransferase mRNAs.

Polyamines regulate gene expression in Escherichia coli by translationally stimulating mRNAs encoding global transcription factors. In this study, we focused on histone acetylation, one of the mechanisms of epigenetic regulation of gene expression, to attempt to clarify the role of polyamines in the regulation of gene expression in eukaryotes. We found that activities of histone acetyltransferases in both the nucleus and cytoplasm decreased significantly in polyamine-reduced mouse mammary carcinoma FM3A cells. Although protein levels of histones H3 and H4 did not change in control and polyamine-reduced cells, acetylation of histones H3 and H4 was greatly decreased in the polyamine-reduced cells. Next, we used control and polyamine-reduced cells to identify histone acetyltransferases whose synthesis is stimulated by polyamines. We found that polyamines stimulate the translation of histone acetyltransferases GCN5 and HAT1. Accordingly, GCN5- and HAT1-catalyzed acetylation of specific lysine residues on histones H3 and H4 was stimulated by polyamines. Consistent with these findings, transcription of genes required for cell proliferation was enhanced by polyamines. These results indicate that polyamines regulate gene expression by enhancing the expression of the histone acetyltransferases GCN5 and HAT1 at the level of translation. Mechanistically, polyamines enhanced the interaction of microRNA-7648-5p (miR-7648-5p) with the 5'-UTR of GCN5 mRNA, resulting in stimulation of translation due to the destabilization of the double-stranded RNA (dsRNA) between the 5'-UTR and the ORF of GCN5 mRNA. Because HAT1 mRNA has a short 5'-UTR, polyamines may enhance initiation complex formation directly on this mRNA.

Translational Regulation of Clock Genes BMAL1 and REV-ERBα by Polyamines.

Polyamines stimulate the synthesis of specific proteins at the level of translation, and the genes encoding these proteins are termed as the "polyamine modulon". The circadian clock generates daily rhythms in mammalian physiology and behavior. We investigated the role of polyamines in the circadian rhythm using control and polyamine-reduced NIH3T3 cells. The intracellular polyamines exhibited a rhythm with a period of about 24 h. In the polyamine-reduced NIH3T3 cells, the circadian period of circadian clock genes was lengthened and the synthesis of BMAL1 and REV-ERBα was significantly reduced at the translation level. Thus, the mechanism of polyamine stimulation of these protein syntheses was analyzed using NIH3T3 cells transiently transfected with genes encoding enhanced green fluorescent protein (EGFP) fusion mRNA with normal or mutated 5'-untranslated region (5'-UTR) of Bmal1 or Rev-erbα mRNA. It was found that polyamines stimulated BMAL1 and REV-ERBα synthesis through the enhancement of ribosomal shunting during the ribosome shunting within the 5'-UTR of mRNAs. Accordingly, the genes encoding Bmal1 and Rev-erbα were identified as the members of "polyamine modulon", and these two proteins are significantly involved in the circadian rhythm control.

Cytotoxic Mechanism of Excess Polyamines Functions through Translational Repression of Specific Proteins Encoded by Polyamine Modulon.

Excessive accumulation of polyamines causes cytotoxicity, including inhibition of cell growth and a decrease in viability. We investigated the mechanism of cytotoxicity caused by spermidine accumulation under various conditions using an Escherichia coli strain deficient in spermidine acetyltransferase (SAT), a key catabolic enzyme in controlling polyamine levels. Due to the excessive accumulation of polyamines by the addition of exogenous spermidine to the growth medium, cell growth and viability were markedly decreased through translational repression of specific proteins [RMF (ribosome modulation factor) and Fis (rRNA transcription factor) etc.] encoded by members of polyamine modulon, which are essential for cell growth and viability. In particular, synthesis of proteins that have unusual locations of the Shine-Dalgarno (SD) sequence in their mRNAs was inhibited. In order to elucidate the molecular mechanism of cytotoxicity by the excessive accumulation of spermidine, the spermidine-dependent structural change of the bulged-out region in the mRNA at the initiation site of the rmf mRNA was examined using NMR analysis. It was suggested that the structure of the mRNA bulged-out region is affected by excess spermidine, so the SD sequence of the rmf mRNA cannot approach initiation codon AUG.

Protective Effects of Brain Infarction by N-Acetylcysteine Derivatives.

BACKGROUND AND PURPOSE: We recently found that acrolein (CH2=CH-CHO) is more strongly involved in brain infarction compared with reactive oxygen species. In this study, we looked for acrolein scavengers with less side effects. METHODS: Photochemically induced thrombosis model mice were prepared by injection of Rose Bengal. Effects of N-acetylcysteine (NAC) derivatives on brain infarction were evaluated using the public domain National Institutes of Health image program. RESULTS: NAC, NAC ethyl ester, and NAC benzyl ester (150 mg/kg) were administered intraperitoneally at the time of induction of ischemia, or these NAC derivatives (50 mg/kg) were administered 3× at 24-h intervals before induction of ischemia and 1 more administration at the time of induction of ischemia. The size of brain infarction decreased in the order NAC benzyl ester>NAC ethyl ester>NAC in both experimental conditions. Detoxification of acrolein occurred through conjugation of acrolein with glutathione, which was catalyzed by glutathione S-transferases, rather than direct conjugation between acrolein and NAC derivatives. The level of glutathione S-transferases at the locus of brain infarction was in the order of administration of NAC benzyl ester>NAC ethyl ester>NAC>no NAC derivatives, suggesting that NAC derivatives stabilize glutathione S-transferases. CONCLUSIONS: The results indicate that detoxification of acrolein by NAC derivatives is caused through glutathione conjugation with acrolein catalyzed by glutathione S-transferases, which can be stabilized by NAC derivatives. This is a new concept of acrolein detoxification by NAC derivatives.

Poly-ion Complex of Chondroitin Sulfate and Spermine and Its Effect on Oral Chondroitin Sulfate Bioavailability.

Chondroitin sulfate (CS) has been accepted as an ingredient in health foods for the treatment of symptoms related to arthritis and cartilage repair. However, CS is poorly absorbed through the gastrointestinal tract because of its high negative electric charges and molecular weight (MW). In this study, poly-ion complex (PIC) formation was found in aqueous solutions through electrostatic interaction between CS and polyamines-organic molecules having two or more primary amino groups ubiquitously distributed in natural products at high concentrations. Characteristic properties of various PICs generated by mixing CS and natural polyamines, including unusual polyamines, were studied based on the turbidity for PIC formation, the dynamic light scattering for the size of PIC particles, and ζ-potential measurements for the surface charges of PIC particles. The efficiency of PIC formation between CS and spermine increased in a CS MW-dependent manner, with 15 kDa CS being critical for the formation of PIC (particle size: 3.41 µm) having nearly neutral surface charge (ζ-potential: -0.80 mV). Comparatively, mixing tetrakis(3-aminopropyl)ammonium and 15 kDa of CS afforded significant levels of PIC (particle size: 0.42±0.16 µm) despite a strongly negative surface charge (-34.67±1.15 mV). Interestingly, the oral absorption efficiency of CS was greatly improved only when PIC possessing neutral surface charges was administered to mice. High formation efficiency and electrically neutral surface charge of PIC particles are important factors for oral CS bioavailability.

Polyamine stimulation of eEF1A synthesis based on the unusual position of a complementary sequence to 18S rRNA in eEF1A mRNA.

It is thought that Shine-Dalgarno-like sequences, which exhibit complementarity to the nucleotide sequences at the 3'-end of 18S rRNA, are not present in eukaryotic mRNAs. However, complementary sequences consisting of more than 5 nucleotides to the 3'-end of 18S rRNA, i.e., a CR sequence, are present at -17 to -32 upstream from the initiation codon AUG in 18 mRNAs involved in protein synthesis except eEF1A mRNA. Thus, effects of the CR sequence in mRNAs and polyamines on protein synthesis were examined using control and polyamine-reduced FM3A and NIH3T3 cells. Polyamines did not stimulate protein synthesis encoded by 18 mRNAs possessing a normal CR sequence. When the CR sequence was deleted, protein synthetic activities decreased to less than 70% of intact mRNAs. In eEF1A mRNA, the CR sequence was located at -33 to -39 upstream from the initiation codon AUG, and polyamines stimulated eEF1A synthesis about threefold. When the CR sequence was shifted to -22 to -28 upstream from the AUG, eEF1A synthesis increased in polyamine-reduced cells and the degree of polyamine stimulation decreased greatly. The results indicate that the CR sequence exists in many eukaryotic mRNAs, and the location of a CR sequence in mRNAs influences polyamine stimulation of protein synthesis.

Properties of putrescine uptake by PotFGHI and PuuP and their physiological significance in Escherichia coli.

Properties of putrescine uptake by PotFGHI and PuuP and their physiological significance were studied using a polyamine biosynthesis and uptake deficient Escherichia coli KK3131 transformed with pACYC184 containing potFGHI or puuP. Putrescine uptake activity of E. coli KK3131 transformed with pACYC184-PotFGHI was higher than that of E. coli 3131 transformed with pACYC-PuuP when cells were cultured in the absence of putrescine. Putrescine uptake by PotFGHI was both ATP and membrane potential dependent, while that by PuuP was membrane potential dependent. Feedback inhibition by polyamines occurred at the PotFGHI uptake system but not at the PuuP uptake system. Expression of PuuP was reduced in the presence of PuuR, a negative regulator for PuuP, and expression of PuuR was positively regulated by glucose, which reduces the level of cAMP. The complex of cAMP and CRP (cAMP receptor protein) inhibited the expression of PuuR in the absence of glucose. Thus, the growth rate of E. coli KK3131 in the presence of both 0.4% (22.2 mM) glucose and 10 mM putrescine was in the order of cells transformed with pACYC-PotFGHI > pACYC-PuuP > pACYC-PuuP + PuuR, which was parallel with the polyamine content in cells. The results indicate that PotFGHI is necessary for rapid cell growth in the presence of glucose as an energy source. When glucose in medium was depleted, however, PuuP was absolutely necessary for cell growth in the presence of putrescine, because accumulation of putrescine to a high level by PuuP was necessary for utilization of putrescine as an energy source.

Caldomycin, a new guanidopolyamine produced by a novel agmatine homocoupling enzyme involved in homospermidine biosynthesis.

An extreme thermophilic bacterium, Thermus thermophilus produces more than 20 unusual polyamines, but their biosynthetic pathways, including homospermidine, are not yet fully understood. Two types of homospermidine synthases have been identified in plants and bacteria, which use spermidine and putrescine or two molecules of putrescine as substrates. However, homospermidine synthases with such substrate specificity have not been identified in T. thermophilus. Here we identified a novel agmatine homocoupling enzyme that is involved in homospermidine biosynthesis in T. thermophilus. The reaction mechanism is different from that of a previously described homospermidine synthase, and involves conjugation of two molecules of agmatine, which produces a diamidino derivative of homospermidine (caldomycin) as an immediate precursor of homospermidine. We conclude that there is a homospermidine biosynthetic pathway from agmatine via caldomycin synthase followed by ureohydrolase in T. thermophilus. Furthermore, it is shown that caldomycin is a novel compound existing in nature.

Inactivation of GAPDH as one mechanism of acrolein toxicity.

We have recently reported that acrolein is more toxic than reactive oxygen species. Thus, the mechanism of cell toxicity by acrolein was studied using mouse mammary carcinoma FM3A cells. Acrolein-conjugated proteins were separated by gel electrophoresis with subsequent determination of their amino acid sequence, and it was found that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was one of the major acrolein-conjugated proteins in cells. Acrolein interacted with cysteine-150 at the active site of GAPDH, and also with cysteine-282. When cells were treated with 8 µM acrolein, the activity of acrolein-conjugated GAPDH was greatly reduced, and the ATP content in cells was thus significantly reduced. In addition, it was shown that acrolein-conjugated GAPDH translocated to the nucleus, and the level of acetylated GAPDH and the number of TUNEL positive cells was increased, indicating that cell death is enhanced by acrolein-conjugated GAPDH. Inhibition of cell growth by acrolein was partially reversed when the cDNA encoding GAPDH was transformed into cells. These results indicate that inactivation of GAPDH is one mechanism that underlies cell toxicity caused by acrolein.

Spermidine is required for morphogenesis in the human pathogenic fungus, Penicillium marneffei.

Penicillium marneffei is a thermally dimorphic fungus that is a highly significant pathogen of immune compromised persons living or having traveled in Southeast Asia. When cultured at 25°C, the wild-type strain of P. marneffei exhibits a mycelial morphology that is marked by the development of specialized structures bearing conidia. Incubation of the wild type at 37°C, however, promotes the development of a yeast form that divides by fission. Development of the yeast morphology in vivo appears to be requisite for pathogenesis. In a prior study using Agrobacterium-mediated transformation for random mutagenesis via T-DNA integration, we generated a morphological mutant (strain I6) defective in conidiation. The T-DNA insertion site in strain I6 was determined to be within the gene encoding S-adenosylmethionine decarboxylase (sadA), an enzyme critical to spermidine biosynthesis. In the present study, we demonstrated that strain I6 was able to grow on rich media in either the mold or yeast forms at 25°C and 37°C, respectively. However, reduced growth of strain I6 was observed on minimal medium at either temperature. In addition, strain I6 produced mycelia with impaired conidiation on minimal medium at 25°C. Supplementation of minimal medium with spermidine restored the ability of strain I6 to produce conidia at 25°C and promoted yeast development at 37°C. Moreover, conidia of strain I6 exhibited poor germination frequencies in the absence of this polyamine. All three of these processes (conidiogenesis, germination, and growth) were reinstated in strain I6 by complementation of the partially deleted of sadA gene by ectopic insertion of an intact wild-type copy. These results augment prior observations that spermidine biosynthesis is essential to normal growth, conidiogenesis, spore germination, and dimorphism in a variety of fungi. Given the presumption that P. marneffei infections are initiated following inhalation of conidia, and that pathogenesis is dependent upon yeast development, this study further suggests that the spermidine biosynthetic pathway may serve as a potential target for combating infections by this medically important fungus.

Urinary Amino Acid-Conjugated Acrolein and Taurine as New Biomarkers for Detection of Dementia.

BACKGROUND: Dementia, including Alzheimer's disease (AD), is one of the serious diseases at advanced age, and its early detection is important for maintaining quality of life (QOL). OBJECTIVE: In this study, we sought novel biomarkers for dementia in urine. METHODS: Samples of urine were collected from 57 control subjects without dementia, 62 mild cognitive impairment (MCI) patients, and 42 AD patients. Mini-Mental State Examination (MMSE) was evaluated when subjects were examined by medical doctors. Urinary amino acid (lysine)-conjugated acrolein (AC-Acro) was measured using N ɛ-(3-formyl-3, 4-dehydropiperidine) lysine (FDP-Lys) ELISA kit, and taurine content was measured using a taurine assay kit. Values were normalized by creatinine content which was measured with the colorimetric assay kit. RESULTS: We found that urinary amino acid (lysine)-conjugated acrolein (AC-Acro) and taurine negatively correlated with MMSE score and are significantly lower in dementia patients compared to the normal subjects. When AC-Acro and taurine were evaluated together with age using an artificial neural network model, median relative risk values for subjects with AD, subjects with mild cognitive impairment, and control subjects were 0.96, 0.53, and 0.06, respectively. CONCLUSION: Since urine is relatively easy to collect, our findings provide a novel biomarker for dementia without invasiveness.

Putrescine Biosynthesis from Agmatine by Arginase (TtARG) in Thermus thermophilus.

In the three domains of life, three biosynthetic pathways are known for putrescine. The first route is conversion of ornithine to putrescine by ornithine decarboxylase (ODC: SpeC), the second route is the conversion of arginine to agmatine by arginine decarboxylase (ADC: SpeA), followed by the conversion of agmatine to putrescine by agmatine ureohydrolase (AUH: SpeB), and the third route is the conversion of agmatine to N-carbamoylputrescine by agmatine deiminase (agmatine iminohydrolase, AIH), followed by the conversion of N-carbamoylputrescine to putrescine by N-carbamoylputrescine amidohydrolase (NCPAH). An extreme thermophile, Thermus thermophilus produces putrescine, although this bacterium lacks homologs for putrescine synthesizing pathways, such as ODC, AUH, AIH and NCPAH. To identify genes involved in putrescine biosynthesis in T. thermophilus, putrescine biosynthesis was examined by disruption of a predicted gene for agmatinase (agmatine ureohydrolase), or by using purified enzyme. It was found that arginase (TTHA1496) showed an agmatinase activity utilizing agmatine as a substrate. These results indicate that this bacterium can use arginase for putrescine biosynthesis. Arginase is a major contributor to putrescine biosynthesis under physiological conditions. The presence of an alternative pathway for converting agmatine into putrescine is functionally important for polyamine metabolism supporting survival at extreme environments.

Augmented glutathione synthesis decreases acrolein toxicity.

We have shown recently that acrolein is more strongly involved in cell damage than reactive oxygen species during brain infarction. Thus, we tried to isolate cells with reduced susceptibility to acrolein toxicity to clarify how acrolein is detoxified under cell culture conditions. The IC(50) of acrolein in mouse mammary carcinoma FM3A cells and in neuroblastoma Neuro2a cells was 2.6 and 4.2µM, respectively, but in acrolein toxicity-decreasing FM3A (FM3A-ATD) cells and Neuro2a (Neuro2a-ATD) cells, it was 7.6 and 8.4µM, respectively. In both FM3A-ATD and Neuro2a-ATD cells, the concentration of glutathione (GSH) was increased, so that detoxification occurred through acrolein conjugation with GSH. In FM3A-ATD cells, the level of a rate-limiting enzyme of GSH synthesis, γ-glutamylcysteine ligase catalytic unit (GCLC), was increased through the reactivation of one inactive allele of GCLC genes in FM3A cells. In Neuro2a-ATD cells, phosphorylation of transcription factors (c-Jun and NF-κB) necessary for expression of genes for GCLC and glutathione synthetase (GSHS) involved in GSH synthesis was stimulated, so that transcription of two genes increased in Neuro2a-ATD cells. Phosphorylation of JNK (c-Jun N-terminal kinase), which catalyzes phosphorylation of c-Jun and NF-κB p65, was also increased in Neuro2a-ATD cells, suggesting that activation of JNK kinase is responsible for the increase in GSH. These results support the idea that GSH plays important roles in detoxification of acrolein, because GSH is increased in both FM3A-ATD and Neuro2a-ATD cells.

Polyamines produced by an extreme thermophile are essential for cell growth at high temperature.

An extreme thermophile, Thermus thermophilus grows at an optimum temperature of around 70°C and produces 16 different polyamines including long-chain and branched-chain polyamines. We found that the composition of polyamines in the thermophile cells changes with culture temperature. Long-chain and branched-chain polyamines (unusual polyamines) were increased in the cells grown at high temperature such as 80°C, but they were minor components in the cells grown at relatively lower temperature such as 60°C. The effects of polyamines on cell growth were studied using T. thermophilus HB8 ΔspeA deficient in arginine decarboxylase. Cell growth of this mutant strain was significantly decreased at 70°C. This mutant strain cannot produce polyamines and grows poorly at 75°C. It was also determined whether polyamines are directly involved in protecting DNA from DNA double-strand breaks (DSBs) induced by heat. Polyamines protected DNA against double-strand breaks. Therefore, polyamines play essential roles in cell growth at extremely high temperature through maintaining a functional conformation of DNA against DSBs and depurination.

Ribosome modulation factor, an important protein for cell viability encoded by the polyamine modulon.

We searched for proteins whose synthesis is enhanced by polyamines at the stationary phase of cell growth using an Escherichia coli polyamine-requiring mutant in which cell viability is greatly decreased by polyamine deficiency. The synthesis of ribosome modulation factor (RMF) was strongly enhanced by polyamines at the level of translation at the stationary phase of cell growth. In rmf mRNA, a Shine-Dalgarno (SD) sequence is located 11 nucleotides upstream of the initiation codon AUG. When the SD sequence was moved to the more common position 8 nucleotides upstream of the initiation codon, the degree of polyamine stimulation was reduced, although the level of RMF synthesis was markedly increased. Polyamine stimulation of RMF synthesis was found to be caused by a selective structural change of the bulged-out region of the initiation site of rmf mRNA. The decrease in cell viability caused by polyamine deficiency was prevented by the addition of a modified rmf gene whose synthesis is not influenced by polyamines. The results indicate that polyamines enhance cell viability of E. coli at least in part by enhancing RMF synthesis.

Identification and functions of amino acid residues in PotB and PotC involved in spermidine uptake activity.

Amino acid residues on PotB and PotC involved in spermidine uptake were identified by random and site-directed mutagenesis. It was found that Trp(8), Tyr(43), Trp(100), Leu(110), and Tyr(261) in PotB and Trp(46), Asp(108), Glu(169), Ser(196), Asp(198), and Asp(199) in PotC were strongly involved in spermidine uptake and that Tyr(160), Glu(172), and Leu(274) in PotB and Tyr(19), Tyr(88), Tyr(148), Glu(160), Leu(195), and Tyr(211) in PotC were moderately involved in spermidine uptake. Among 11 amino acid residues that were strongly involved in spermidine uptake, Trp(8) in PotB was important for insertion of PotB and PotC into membranes. Tyr(43), Trp(100), and Leu(110) in PotB and Trp(46), Asp(108), Ser(196), and Asp(198) in PotC were found to be involved in the interaction with PotD. Leu(110) and Tyr(261) in PotB and Asp(108), Asp(198), and Asp(199) in PotC were involved in the recognition of spermidine, and Trp(100) and Tyr(261) in PotB and Asp(108), Glu(169), and Asp(198) in PotC were involved in ATPase activity of PotA. Accordingly, Trp(100) in PotB was involved in both PotD recognition and ATPase activity, Leu(110) in PotB was involved in both PotD and spermidine recognition, and Tyr(261) in PotB was involved in both spermidine recognition and ATPase activity. Asp(108) and Asp(198) in PotC were involved in PotD and spermidine recognition as well as ATPase activity. These results suggest that spermidine passage from PotD to the cytoplasm is coupled to the ATPase activity of PotA through a structural change of PotA by its ATPase activity.

Calditerricola satsumensis gen. nov., sp. nov. and Calditerricola yamamurae sp. nov., extreme thermophiles isolated from a high-temperature compost.

Two novel thermophilic micro-organisms, designated YMO81(T) and YMO722(T), were isolated from a high-temperature compost (internal temperature > 95 °C). The isolates were able to grow at 80 °C in a nutrient broth and in a synthetic medium. Cells were aerobic, Gram-negative rods (0.3 x 4.0 µm). Spore formation was not observed. Strain YMO81(T) grew at 83 °C and pH 6.9-8.9 and grew optimally at 78 °C and pH 7.5 with 2  % NaCl. For growth in a synthetic minimal medium at 70 °C, the vitamins biotin, folic acid and thiamine and the amino acids glutamine and methionine were essential for growth of both strains; at 80 °C, strain YMO81(T) also required histidine, isoleucine, leucine, lysine, phenylalanine, serine, tryptophan and valine. Cellular fatty acids of the isolates comprised mainly iso-C17:0 and anteiso-C17:0. The DNA G+C contents of strains YMO81(T) and YMO722(T) were 70 and 64 mol%, respectively. When the 16S rRNA gene sequences of the isolates were compared with those of other bacteria, highest similarity was observed with Planifilum yunnanense LA5(T) (90 % 16S rRNA gene sequence similarity). DNA-DNA relatedness between strain YMO722(T) and strain YMO81(T) was 55  %. N4-Aminopropylspermine was identified as a major polyamine, which suggested that the isolates were distinct from other related taxa. On the basis of phylogenetic, phenotypic and chemotaxonomic analyses, we propose a new genus, Calditerricola gen. nov., and two novel species, the type species Calditerricola satsumensis sp. nov., with type strain YMO81(T) (=ATCC BAA-1462(T) =JCM 14719(T) =DSM 45223(T)), and Calditerricola yamamurae sp. nov., with type strain YMO722(T) (=ATCC BAA-1461(T) =JCM 14720(T) =DSM 45224(T)).