Validation Study of LuciPacTM A3 Surface for Hygiene Monitoring through Detection of ATP, ADP, and AMP from Stainless Steel Surfaces: AOAC Performance Tested MethodSM 051901.

BACKGROUND: The LuciPac A3 Surface Hygiene Monitoring System based on the detection of total adenylate, ATP+ADP+AMP (A3), has been developed by Kikkoman Biochemifa. OBJECTIVE: This A3 swabbing assay kit was validated for Performance Tested MethodsSM (PTM) certification. METHODS: The LuciPac A3 Surface Hygiene Monitoring System was evaluated for limit of detection (LOD) for each adenylate with pure analyte solutions, detection of food residues and microbial residues on stainless steel surfaces, interference by disinfectants, and selectivity of the method response. RESULTS: Pure analyte studies performed by the method developer and the independent laboratory showed good linearity (R2 > 0.9854) and repeatability precision (RSDr < 20% for ≥2.5 fmol/assay). The LOD values for each adenylate were around 10 relative light units or 2.5 fmol/assay. The repeatability precision in the method developer laboratory for the matrix study (raw chicken breast, sliced deli ham, orange juice, yogurt, and apple pie) and the microbial study (Cronobacter sakazakii, Lactobacillus acidophilus, and Saccharomyces cerevisiae) were 8-30% and 10-35%, respectively. The repeatability precision of independent laboratory testing was 13-27% for orange juice and 16-43% for ham. Interference by several disinfectants indicate that rinsing is recommended to be performed after the use of sanitizing agents and before testing with LuciPac A3 Surface. Selectivity testing revealed that no positive interference and no inhibition were caused by adenylate analogues. Instrument variation, lot-to-lot consistency, accelerated stability (30°C, 5 weeks) were confirmed, and the method was shown to be robust against shaking time. CONCLUSIONS: The LuciPac A3 Surface has been successfully validated. HIGHLIGHTS: This A3 swabbing assay kit was qualified for PTM certification No. 051901.

Vestiges of Adaptation to the Mesophilic Environment in the Genome of Tepiditoga spiralis gen. nov., sp. nov.

A novel anaerobic heterotrophic strain, designated strain sy52T, was isolated from a hydrothermal chimney at Suiyo Seamount in the Pacific Ocean. A 16S rRNA gene analysis revealed that the strain belonged to the family Petrotogaceae in the phylum Thermotogae. The strain was mesophilic with optimum growth at 48°C and the phylum primarily comprised hyperthermophiles and thermophiles. Strain sy52T possessed unique genomic characteristics, such as an extremely low G+C content and 6 copies of rRNA operons. Genomic analyses of strain sy52T revealed that amino acid usage in the predicted proteins resulted from adjustments to mesophilic environments. Genomic features also indicated independent adaptions to the mesophilic environment of strain sy52T and Mesotoga species, which belong to the mesophilic lineage in the phylum Thermotogae. Based on phenotypic and phylogenetic evidence, strain sy52T is considered to represent a novel genus and species in the family Petrotogaceae with the proposed name Tepiditoga spiralis gen. nov., sp. nov.

Diversity of PKS and NRPS gene clusters between Streptomyces abyssomicinicus sp. nov. and its taxonomic neighbor.

Streptomyces sp. CHI39, isolated from a rock soil sample, is a producer of abyssomicin I. The taxonomic status was clarified by a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences showed that the strain was closely related to Streptomyces fragilis, with similarity of 99.9%. Strain CHI39 comprised LL-diaminopimelic acid, glutamic acid, glycine, and alanine in its peptidoglycan. The predominant menaquinones were MK-9(H6), and major fatty acids were anteiso-C15:0, anteiso-C17:0, and iso-C16:0. The chemotaxonomic features matched those described for the genus Streptomyces. Genome sequencing was conducted for strain CHI39 and S. fragilis NBRC 12862T. The results of digital DNA-DNA hybridization along with differences in phenotypic characteristics between the strains suggested strain CHI39 to be a novel species, for which Streptomyces abyssomicinicus sp. nov. is proposed; the type strain is CHI39T (=NBRC 110469T). Next, we surveyed polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) gene clusters in genomes of S. abyssomicinicus CHI39T and S. fragilis NBRC 12862T. These strains encoded 9 and 12 clusters, respectively, among which only four clusters were shared between them while the others are specific in each strain. This suggests that strains classified to distinct species each harbor many specific secondary metabolite-biosynthetic pathways even if the strains are taxonomically close.

Gene Mutations in Ganoderma lucidum During Long-Term Preservation by Repeated Subculturing.

Subculturing is frequently used for the preservation of basidiomycetes. In this study, to assess and verify the risks of repeated subculturing on the long-term preservation of strains of culture collections, we performed single nucleotide polymorphism (SNP) analysis in genes encoding enzymes of the mevalonate pathway, 1,3-ß-glucan synthesis, lignin degradation, and the tricarboxylic acid (TCA) cycle of mycelia before and after preserving for a 4-year period by the subculturing 30 times every 45 days of Ganoderma lucidum NBRC 8346. As a result of analyzing 60 genes of the strain, SNPs were found in 18 genes, and 14 of them were found in the exon region. Nonsynonymous coding SNPs were found in two genes (atoB_2, hmgr) encoding enzymes of mevalonate pathway and five genes (lcc1_9, lcc1_11, lcc1_13, dslcc6, aa5_1_9) encoding enzymes of lignin degradation after subculturing of G. lucidum NBRC 8346.

Diversity of nonribosomal peptide synthetase and polyketide synthase gene clusters among taxonomically close Streptomyces strains.

To identify the species of butyrolactol-producing Streptomyces strain TP-A0882, whole genome-sequencing of three type strains in a close taxonomic relationship was performed. In silico DNA-DNA hybridization using the genome sequences suggested that Streptomyces sp. TP-A0882 is classified as Streptomyces diastaticus subsp. ardesiacus. Strain TP-A0882, S. diastaticus subsp. ardesiacus NBRC 15402T, Streptomyces coelicoflavus NBRC 15399T, and Streptomyces rubrogriseus NBRC 15455T harbor at least 14, 14, 10, and 12 biosynthetic gene clusters (BGCs), respectively, coding for nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). All 14 gene clusters were shared by S. diastaticus subsp. ardesiacus strains TP-A0882 and NBRC 15402T, while only four gene clusters were shared by the three distinct species. Although BGCs for bacteriocin, ectoine, indole, melanine, siderophores such as deferrioxamine, terpenes such as albaflavenone, hopene, carotenoid and geosmin are shared by the three species, many BGCs for secondary metabolites such as butyrolactone, lantipeptides, oligosaccharide, some terpenes are species-specific. These results indicate the possibility that strains belonging to the same species possess the same set of secondary metabolite-biosynthetic pathways, whereas strains belonging to distinct species have species-specific pathways, in addition to some common pathways, even if the strains are taxonomically close.

Genetic variation during long-term preservation of bacteria in public culture collections.

Phenotypic and genetic changes during long-term preservation have been observed in microbial strains at culture collections (CCs). It is imperative to verify the effects of these changes on quality of the strains preserved at CCs. In this study, we performed genome-wide single-nucleotide polymorphism (SNP) analysis of different production lots, which had been derived from the same origin and preserved at the NITE Biological Resource Center (NBRC) for a 4-38-year period by the vacuum liquid drying method at 4 °C. The analysis was conducted for three sets of lots derived from Cellulomonas fimi NBRC 15513T, Corynebacterium glutamicum NBRC 12168T, and Saccharomonospora viridis NBRC 12207T. SNPs were found in all sets studied for comparison purposes. In sets of two or three lots, genomic SNPs were found in both non-coding sequences (non-CDSs) and in coding sequences (CDSs), and the SNPs in the CDSs resulted in non-synonymous mutations. These data indicated that genomic variation occurred during long-term preservation.

Galvanic Tongue Stimulation Inhibits Five Basic Tastes Induced by Aqueous Electrolyte Solutions.

Galvanic tongue stimulation (GTS) modulates taste sensation. However, the effect of GTS is contingent on the electrode polarity in the proximity of the tongue. If an anodal electrode is attached in the proximity of the tongue, an electrical or metallic taste is elicited. On the other hand, if only cathodal electrode is attached in the proximity of the tongue, the salty taste, which is induced by electrolyte materials, is inhibited. The mechanism of this taste inhibition is not adequately understood. In this study, we aim to demonstrate that the inhibition is cause by ions, which elicit taste and which migrate from the taste sensors on the tongue by GTS. We verified the inhibitory effect of GTS on all five basic tastes induced by electrolyte materials. This technology is effective for virtual reality systems and interfaces to support dietary restrictions. Our findings demonstrate that cathodal-GTS inhibits all the five basic tastes. The results also support our hypothesis that the effects of cathodal-GTS are caused by migrating tasting ions in the mouth.

Reclassification of Amycolicicoccus subflavus as Hoyosella subflava comb. nov. and emended descriptions of the genus Hoyosella and Hoyosella altamirensis.

16S rRNA gene sequences of two type strains belonging to different genera within the suborder Corynebacterineae, namely Hoyosella altamirensis and Amycolicicoccus subflavus, show a similarity of 99.8 %. Therefore, in order to clarify their taxonomic relationship, a polyphasic recharacterization under the same conditions was carried out. The peptidoglycan of H. altamirensis NBRC 109631T and A. subflavus NBRC 109087T was of A1γ type with meso-diaminopimelic acid as their diagnostic diamino acid. Both strains contained MK-8 as the only detected menaquinone, C16 : 0 and C18 : 1ω9c as the major fatty acids, and diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol as the principal polar lipids. The coincidences of these chemotaxonomic features suggested that H. altamirensis and A. subflavus should be assigned to the same genus. Meanwhile, the average nucleotide identity value between both strains and the results of physiological and biochemical tests indicated that H. altamirensis and A. subflavus should be affiliated to different species. Therefore, according to Rules 38 and 41a of the Bacteriological Code, it is proposed that Amycolicicoccus subflavus Wang et al. 2010 be reclassified as Hoyosella subflava comb. nov. (type strain DQS3-9A1T=CGMCC 4.3532T=DSM 45089T=JCM 17490T=NBRC 109087T) and the descriptions of the genus HoyosellaJurado et al. 2009 and Hoyosella altamirensisJurado et al. 2009 are emended accordingly.

Subcellular distribution of central carbohydrate metabolism pathways in the red alga Cyanidioschyzon merolae.

MAIN CONCLUSION: Comprehensive subcellular localization analysis revealed that the subcellular distribution of carbohydrate metabolic pathways in the red alga Cyanidioschyzon is essentially identical with that in Arabidopsis , except the lack of transaldolase. In plants, the glycolysis and oxidative pentose phosphate pathways (oxPPP) are located in both cytosol and plastids. However, in algae, particularly red algae, the subcellular localization of enzymes involved in carbon metabolism is unclear. Here, we identified and examined the localization of enzymes related to glycolysis, oxPPP, and tricarboxylic acid (TCA) and Calvin-Benson cycles in the red alga Cyanidioschyzon merolae. A gene encoding transaldolase of the oxPPP was not found in the C. merolae genome, and no transaldolase activity was detected in cellular extracts. The subcellular localization of 65 carbon metabolic enzymes tagged with green fluorescent protein or hemagglutinin was examined in C. merolae cells. As expected, TCA and Calvin-Benson cycle enzymes were localized to mitochondria and plastids, respectively. The analyses also revealed that the cytosol contains the entire glycolytic pathway and partial oxPPP, whereas the plastid contains a partial glycolytic pathway and complete oxPPP, with the exception of transaldolase. Together, these results suggest that the subcellular distribution of carbohydrate metabolic pathways in C. merolae is essentially identical with that reported in the photosynthetic tissue of Arabidopsis thaliana; however, it appears that substrates typically utilized by transaldolase are consumed by glycolytic enzymes in the plastidic oxPPP of C. merolae.

Detection and characterization of phosphatidylcholine in various strains of the genus Chlamydomonas (Volvocales, Chlorophyceae).

The laboratory strains of Chlamydomonas reinhardtii have been reported to contain no phosphatidylcholine (PC), which is considered to be replaced by another zwitterionic lipid, diacylglyceryl-N,N,N-trimethylhomoserine (DGTS). According to the recently published classification, the strains belonged to the subgroup Reinhardtinia. Screening for PC in 13 selected strains of Chlamydomonas in the NIES Algal Collection, which are different in habitats and belong to different phylogenetic subgroups in the genus, revealed the presence of PC in four strains: a strain in the subgroup Polytominia, and three strains in Reinhardtinia. PC was not detected in three other strains of Reinhardtinia analyzed. The presence/absence of PC was not related to the phylogenetic relationship based on 18S rRNA. DGTS was detected in all the strains analyzed. The rare isomer of linolenic acid, 18:3(5,9,12), which has been found in the DGTS of C. reinhardtii, was found in the PC of the two strains and in the DGTS of the five strains. The occurrence of this fatty acid seems limited to a branch of Reinhardtinia. Acquisition and loss of PC in various strains of Chlamydomonas are discussed from the viewpoint of evolution of PC biosynthetic pathway.

Detailed identification of fatty acid isomers sheds light on the probable precursors of triacylglycerol accumulation in photoautotrophically grown Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii is a model alga for studying triacylglycerol (TAG) accumulation in the photosynthetic production of biofuel. Previous studies were conducted under photoheterotrophic growth conditions in medium supplemented with acetate and/or ammonium. We wanted to demonstrate TAG accumulation under truly photoautotrophic conditions without reduced elements. We first reidentified all lipid components and fatty acids by mass spectrometry, because the currently used identification knowledge relies on data obtained in the 1980s. Accordingly, various isomers of fatty acids, which are potentially useful in tracing the flow of fatty acids leading to the accumulation of TAG, were detected. In strain CC1010 grown under photoautotrophic conditions, TAG accumulated to about 57.5 mol% of total lipids on a mole fatty acid basis after the transfer to nitrogen-deficient conditions. The content of monogalactosyl diacylglycerol, sulfoquinovosyl diacylglycerol, and phosphatidylglycerol decreased drastically. The accumulated TAG contained 16:0 as the major acid and 16:4(4,7,10,13), 18:2(9,12), and 18:3(9,12,15), which are typically found in chloroplast lipids. Additionally, 18:1(11) and 18:3(5,9,12), which are specific to extrachloroplast lipids, were also abundant in the accumulated TAG. Photosynthesis and respiration slowed markedly after the shift to nitrogen-deficient conditions. These results suggest that fatty acids for the production of TAG were supplied not only from chloroplast lipids but also from other membranes within the cells, although the possibility of de novo synthesis cannot be excluded. Under nitrogen-replete conditions, supplementation with a high concentration of CO2 promoted TAG production in the cells grown photoautotrophically, opening up the possibility to the continuous production of TAG using CO2 produced by industry.

Role of the glyoxylate pathway in acetic acid production by Acetobacter aceti.

Wild-type Acetobacter aceti NBRC 14818 possesses genes encoding isocitrate lyase (aceA) and malate synthase (glcB), which constitute the glyoxylate pathway. In contrast, several acetic acid bacteria that are utilized for vinegar production lack these genes. Here, an aceA-glcB knockout mutant of NBRC 14818 was constructed and used for investigating the role of the glyoxylate pathway in acetate productivity. In medium containing ethanol as a carbon source, the mutant grew normally during ethanol oxidation to acetate, but exhibited slower growth than that of the wild-type strain as the accumulated acetate was oxidized. The mutant grew similarly to that of the wild-type strain in medium containing glucose as a carbon source, indicating that the glyoxylate pathway was not necessary for glucose utilization. However, in medium containing both ethanol and glucose, the mutant exhibited significantly poorer growth and lower glucose consumption compared to the wild-type strain. Notably, the mutant oxidized ethanol nearly stoichiometrically to acetate, which was retained in the medium for a longer period of time than the acetate produced by wild-type strain. The features of the aceA-glcB knockout mutant revealed here indicate that the lack of the glyoxylate pathway is advantageous for industrial vinegar production by A. aceti.

Changes in the gene expression profile of Acetobacter aceti during growth on ethanol.

Acetobacter aceti NBRC 14818 shows a diauxic growth profile and temporarily accumulates acetate when grown in medium containing ethanol. However, the mechanisms underlying the metabolic switching between the incomplete oxidation of ethanol and overoxidation of acetate, and the control of stress resistance systems in A. aceti cells grown on ethanol are not fully understood. In this study, time-dependent transcriptome changes in cells during growth on ethanol were analyzed by DNA microarray. In A. aceti, ethanol is oxidized to acetate via acetaldehyde by sequential reactions of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). We found that the genes encoding pyrroloquinoline quinone-dependent ADH, membrane-bound ALDH, and two NAD(+)-ADHs were expressed constitutively in cells throughout the culture period. In contrast, the expression levels of genes encoding tricarboxylic acid (TCA) cycle enzymes were low during acetate accumulation until ethanol was consumed, but were significantly upregulated after the accumulated acetate was started to be consumed. This result suggests that changes in the carbon metabolic flow through the TCA cycle are important for the metabolic switching from acetate accumulation to the overoxidation of acetate. In addition, the genes for glyoxylate pathway enzymes were significantly upregulated soon after the cells began oxidizing ethanol, indicating that this pathway is important for the utilization of ethanol as a carbon source.

Transcriptome response to different carbon sources in Acetobacter aceti.

The draft genome sequence of Acetobacter aceti NBRC 14818 was determined by whole-genome shotgun sequencing and the transcriptome profile in cells exponentially grown on ethanol, acetate or glucose was analysed by using a DNA microarray. The genes for all enzymes that constitute the complete tricarboxylic acid (TCA) cycle and glyoxylate pathway were identified in the genome. The TCA cycle genes showed higher expression levels in A. aceti cells grown on acetate or glucose and the glyoxylate pathway genes were significantly induced by ethanol or acetate. Many SOS-response genes were upregulated in cells grown on ethanol, indicating that ethanol provoked damage of DNA and proteins. The superoxide dismutase and catalase genes showed high expression levels in culture on glucose, indicating that oxidation of glucose induced oxidative stress. A. aceti NBRC 14818 was found to have a highly branched respiratory chain. The genes for two type I and one type II NADH dehydrogenase were identified. The genes for one of the type I enzymes were highly expressed when cells were grown on acetate or glucose, but were significantly downregulated in culture on ethanol, probably because ubiquinones were directly reduced by pyrroloquinoline quinone-dependent alcohol dehydrogenase. Four sets of the genes for quinol oxidases, one bo(3)-type (BO3), one bd-type and two cyanide-insensitive-types (CIOs), were identified in the genome. The genes for BO3, which might have proton-pumping activity, were highly expressed under the conditions tested, but were downregulated in the glucose culture. In contrast, the genes for one of the CIOs were significantly upregulated in cells grown on glucose. The two CIOs, which are expected to have lower energy-coupling efficiency, seemed to have a higher contribution in glucose-grown cells. These results indicate that energy conservation efficiency is fine-tuned by changing the respiratory components according to the growth conditions in A. aceti cells.