Takashi Yamazaki

Abstract The control of microbes in manned spaceflight is essential to reducing the risk of infection and maintaining crew health. The primary issue is ensuring the safety of a potable water system, where simultaneous monitoring of microbial abundance and community structure is needed. In this paper, we develop a flow cytometry-based counting protocol targeting cellular flavin autofluorescence as a tool for rapid monitoring of bacterial cells in water. This was successfully applied to estimate the bacterial bioburden in the potable water collected from the International Space Station. We also demonstrate the efficacy of the MinION nanopore sequencer in rapidly characterizing bacterial community structure and identifying the dominant species. These monitoring protocols' rapidity and cost effectiveness would contribute to developing sustainable real-time surveillance of potable water in spaceflight.

<title>Abstract</title> Analysis of the skin mycobiome of an astronaut during a 1-year stay on the International Space Station (ISS) revealed an increased relative abundance of Malassezia restricta and level of Malassezia colonization, and the presence of Cyberlindnera jadinii and Candida boidinii, uncommon skin mycobiome taxa. Similar observations were made in astronauts during a 6-month stay on the ISS (Med Mycol. 2016; 54: 232–239). Future plans for extended space travel should consider the effect of high levels of Malassezia colonization over long periods on astronauts’ skin, and the abnormal proliferation of uncommon microorganisms that may occur in closed environments such as the ISS.

Summary Roots of land plants show gravitropism and hydrotropism in response to gravity and moisture gradients, respectively, for controlling their growth orientation. Gravitropism interferes with hydrotropism, although the mechanistic aspects are poorly understood. Here, we differentiated hydrotropism from gravitropism in cucumber roots by conducting clinorotation and spaceflight experiments. We also compared mechanisms regulating hydrotropism and auxin‐regulated gravitropism. Clinorotated or microgravity (μG)‐grown cucumber seedling roots hydrotropically bent toward wet substrate in the presence of moisture gradients, but they grew straight in the direction of normal gravitational force at the Earth's surface (1G) on the ground or centrifuge‐generated 1G in space. The roots appeared to become hydrotropically more sensitive to moisture gradients under μG conditions in space. Auxin transport inhibitors significantly reduced the hydrotropic response of clinorotated seedling roots. The auxin efflux protein CsPIN5 was differentially expressed in roots of both clinorotated and μG‐grown seedlings; with higher expression in the high‐humidity (concave) side than the low‐humidity (convex) side of hydrotropically responding roots. Our results suggest that roots become hydrotropically sensitive in μG, and CsPIN5‐mediated auxin transport has an important role in inducing root hydrotropism. Thus, hydrotropic and gravitropic responses in cucumber roots may compete via differential auxin dynamics established in response to moisture gradients and gravity.

We investigated the effects of microgravity environment on growth and plant hormone levels in dark‐grown rice shoots cultivated in artificial 1 g and microgravity conditions on the International Space Station (ISS). Growth of microgravity‐grown shoots was comparable to that of 1 g‐grown shoots. Endogenous levels of indole‐3‐acetic acid (IAA) in shoots remained constant, while those of abscisic acid (ABA), jasmonic acid (JA), cytokinins (CKs) and gibberellins (GAs) decreased during the cultivation period under both conditions. The levels of auxin, ABA, JA, CKs and GAs in rice shoots grown under microgravity conditions were comparable to those under 1 g conditions. These results suggest microgravity environment in space had minimal impact on levels of these plant hormones in rice shoots, which may be the cause of the persistence of normal growth of shoots under microgravity conditions. Concerning ethylene, the expression level of a gene for 1‐aminocyclopropane‐1‐carboxylic acid (ACC) synthase, the key enzyme in ethylene biosynthesis, was reduced under microgravity conditions, suggesting that microgravity may affect the ethylene production. Therefore, ethylene production may be responsive to alterations of the gravitational force.

Abstract Reorientation of cucumber seedlings induces re-localization of CsPIN1 auxin efflux carriers in endodermal cells of the transition zone between hypocotyl and roots. This study examined whether the re-localization of CsPIN1 was due to the graviresponse. Immunohistochemical analysis indicated that, when cucumber seedlings were grown entirely under microgravity conditions in space, CsPIN1 in endodermal cells was mainly localized to the cell side parallel to the minor axis of the elliptic cross-section of the transition zone. However, when cucumber seeds were germinated in microgravity for 24 h and then exposed to 1g centrifugation in a direction crosswise to the seedling axis for 2 h in space, CsPIN1 was re-localized to the bottom of endodermal cells of the transition zone. These results reveal that the localization of CsPIN1 in endodermal cells changes in response to gravity. Furthermore, our results suggest that the endodermal cell layer becomes a canal by which auxin is laterally transported from the upper to the lower flank in response to gravity. The graviresponse-regulated re-localization of CsPIN1 could be responsible for the decrease in auxin level, and thus for the suppression of peg formation, on the upper side of the transition zone in horizontally placed seedlings of cucumber.

ABSTRACT As a part of a series of studies regarding the microbial biota in manned space environments, fungi were isolated from six pieces of equipment recovered from the Japanese Experimental Module “KIBO” of the International Space Station and from a space shuttle. Thirty‐seven strains of fungi were isolated, identified and investigated with regard to morphological phenotypes and antifungal susceptibilities. The variety of fungi isolated in this study was similar to that of several previous reports. The dominant species belonged to the genera Penicillium, Aspergillus and Cladosporium, which are potential causative agents of allergy and opportunistic infections. The morphological phenotypes and antifungal susceptibilities of the strains isolated from space environments were not significantly different from those of reference strains on Earth.

The International Space Station (ISS) is a completely closed environment that offers a long-term microgravity environment. It is a unique environment where microbes can fly and attach themselves to devices or humans, especially the exposed parts of the body and head. The ongoing monitoring and analysis of microbes and their movement inside the Japanese Experiment Module (named “Kibo”) of the ISS are intended to study the effects of microbes on humans and prevent health hazards caused by microbes during a long-term space mission. This paper describes the current status and future plan of Japanese microbiological experiments to monitor microbial dynamics in Kibo. It also describes the future prospective and prioritized microbiological research areas based on the “Kibo utilization scenario towards 2020 in the field of life science.” Given the microbial research in space being actively conducted by the USA, NASA and international activities are also reported.

We isolated a target gene for the Lentinula edodes putative transcription factor Le.CDC5 that contains a c-Myb-type DNA-binding domain. The gene, termed ctg1, encodes a novel protein (159 amino acid residues) with a leucine zipper-like sequence and contains a 7-bp Le.CDC5-binding sequence, 5'GCAATCT3', in its transcribed region downstream of the start codon. Chromatin immunoprecipitation analysis strongly suggested that intracellular Le.CDC5 binds to this 7-bp sequence on L. edodes chromatin. Binding was most efficient on chromatin from the stipes of mature fruiting bodies. Two Le.CDC5-interaction partners were identified in L. edodes and named CIPA and CIPB. The CIPB protein (127 amino acid residues) binds to a 6-bp sequence with the consensus sequence 5'CAACAC/T/G3'. The ctg1 gene contains nine 6-bp consensus (or consensus-like) sequences, six are in the 5'-upstream region and three in the transcribed region downstream of the start codon. At least two each of the upstream and downstream sequences appear to bind CIPB in vitro. We suggest that Le.CDC5 and CIPB can cooperatively regulate the expression of ctg1. (c) 2008 Elsevier Inc. All rights reserved.

We cloned and sequenced a photoreceptor gene (Le.phrA) from the basidiomycete Lentinula edodes. The product of Le.phrA, Le.PHRA (924 aa residues) contained a serine-rich region, an LOV domain and two PAS domains. It was clearly smaller than other fungal LOV domain-containing blue-light photoreceptors such as Coprinopsis cinerea Dst1 (1,175 aa), Neurospora crassa WC-1 (1,167 aa), and Cryptococcus neoformans WC-1 (1,141 aa). The Le.phrA gene was found to be transcribed at all stages of the fruiting-body formation of L. edodes, but it was most abundantly transcribed in the immature fruiting body. Fully-matured fruiting body also contained relatively large amounts of Le.phrA transcript. Although the transcript level was lower, preprimordial aggregated mycelial cells grown under a light environment contained larger amounts of the transcript than those grown under continuous darkness, suggesting a light-enhanced expression of the Le.phrA gene. Hymenophore-depleted pileus contained a markedly higher level of the transcript than the stipe.

Recombinants were generated from the ectomycorrhizal basidiomycete, Suillus grevillei, through agroinfection using a binary vector carrying the hygromycin B resistance and the autofluorescent protein, DsRed2, markers. DsRed2 was driven by a cis-regulatory region of the glyceraldeyde-3-phosphate dehydrogenase gene (gpd) from the wood-rotting basidiomycete, Coriolus hirsutus, which contains promoters and 5' gpd sequences with first through fourth exons and expressed for the first time in Suillus spp. The transformation system and recombinants expressing an autofluorescent protein may be useful in genetic analysis of the symbiosis.

The homobasidiomycete Coriolus hirsutus coding sequences of a lignin peroxidase (LiP) gene (lip, containing six (I–VI) introns), a lip cDNA (lipc), and three lipc derivatives containing one (I), three (I–III), or five (I–V) introns were inserted into chromosome-integrating expression vector. These recombinant plasmids were introduced into C. hirustus monokaryotic strain. The transformant carrying the promoter–lipc–terminator cassette did not contain enough mRNA molecules to be detectable by Northern-blot analysis. On the other hand, all the transformants carrying cassettes of genomic lip and intron(s)-containing lipc sequences contained sufficient amounts of mRNAs to be easily detected by Northern-blot analysis. LiP activities in the culture supernatants of these transformants were found to be about five times as high as those of transformants carrying the lipc cassette (or no cassette). The culture supernatants of the transformants with high LiP activity showed remarkably high conversion activity toward pentachlorophenol (PCP) and degradation activity toward 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD). These results indicate that at least one intron (intron I) is required for accumulation of lip mRNA and its subsequent translational expression in C. hirsutus.

We cloned and sequenced a recQ gene homolog from the basidiomycetous mushroom Lentinula edodes (Le.). This gene, named Le.recQ, was found to have a coding capacity of 945 amino acids (aa) and be interrupted by 11 small introns. The deduced Le.RECQ protein was clearly smaller than other fungal RecQ proteins such as Neurospora crassa QDE3 (1955 aa), Schizosaccharomyces pombe Rqh1 (1328 aa), and Saccharomyces cerevisiae SGS1 (1447 aa). It exhibited the highest homology to the Arabidopsis thaliana RecQ14A protein (1182 aa) in its size and as sequence. Northern-blot analysis showed that the Le.recQ gene is transcribed at similar levels during mycelial development in L. edodes fruiting-body formation on a sawdust-corn bran medium. The L. edodes dikaryotic mycelial cells, which had been vegetatively grown in SMY liquid medium, were found to contain a clearly larger amount of Le.recQ transcript than the L. edodes two compatible monokaryotic mycelial cells. Expression of Le.recQ cDNA in S. cerevisiae might partially complement defects associated with the loss of its homolog S. cerevisiae SGS1 gene.

A cDNA homologue of Schizosaccharomyces pombe cdc5(+) was isolated from the basidiomycete mushroom Lentinula edodes and it was named Le.cdc5 cDNA. The deduced Le.CDC5 (842 amino acid residues) possessed N-terminal amino acid sequence highly homologous to those of S. pombe cdc5(+) gene product (Sp.cdc5p) and Sp.cdc5p-related proteins (SPCDC5RPs). The N-terminal 185 amino acid peptide of Le.CDC5 (Le.CDC5(1-185) peptide) produced in Escherichia coli was subjected to random binding-site selection analysis, revealing that Le.CDC5(1-185) peptide binds to a 7-bp sequence with the consensus sequence of 5' GCAATGT3' (complementary; 5' ACATTGC3'). Genomic binding-site (GBS) cloning by using Le.CDC5(1-185) peptide resulted in an isolation of the DNA fragment that contained three sets of 7-bp consensus-like sequence and TATA box. The Le.CDC5 protein contained two putative phosphorylation sites of cAMP-dependent protein kinase (A kinase) in its C-terminus. There exists a possible leucine zipper between the two phosphorylation sites. The Le.CDC5 fragment containing the two phosphorylation sites was actually phosphorylated by commercially available A kinase. Yeast two-hybrid analysis suggested the homodimerization of Le.CDC5 protein probably through the leucine zipper. Northern blot analysis showed that Le.cdc5 gene is most actively transcribed in primordia and small immature fruiting bodies of L. edodes, implying that Le.cdc5 may play a role in the beginning and early stage of fruiting-body formation. (C) 2004 Elsevier B.V. All rights reserved.

Using the method of genomic binding-site cloning, we identified three target genes of the developmental regulator, the product of priB gene (PRIB) in Lentinula edodes: the previously cloned priB and uck1 (UMP-CMP kinase gene) and a new gene, which we named mfbC. Identification of the former two genes was expected, because the promoter regions of priB and the gene encoding UMP-CMP kinase (uck1) have been shown to contain four or two consensus-like sequences of PRIB binding respectively. The mfbC gene contained two consensus-like sequences of PRIB binding in its promoter region and the PRIB protein bound them. The deduced 330 amino acid sequence of the product of mfbC gene (MFBC) was highly homologous to the 325 amino acid sequence of S. cerevisiae YJR070C/Lia1, the protein interacting with a putative translation initiation factor. Only the mature fruiting body of L. edodes was shown to contain the transcript of the mfbC gene almost exclusively, suggesting that mfbC may play a role in the final stage of fruiting-body formation.