ATG and ESCRT control multiple modes of microautophagy.

The discovery of microautophagy, the direct engulfment of cytoplasmic material by the lysosome, dates back to 1966 in a morphological study of mammalian cells by Christian de Duve. Since then, studies on microautophagy have shifted toward the elucidation of the physiological significance of the process. However, in contrast to macroautophagy, studies on the molecular mechanisms of microautophagy have been limited. Only recent studies revealed that ATG proteins involved in macroautophagy are also operative in several types of microautophagy and that ESCRT proteins, responsible for the multivesicular body pathway, play a central role in most microautophagy processes. In this review, we summarize our current knowledge on the function of ATG and ESCRT proteins in microautophagy.

Metabolism-linked methylotaxis sensors responsible for plant colonization in Methylobacterium aquaticum strain 22A.

Motile bacteria take a competitive advantage in colonization of plant surfaces to establish beneficial associations that eventually support plant health. Plant exudates serve not only as primary growth substrates for bacteria but also as bacterial chemotaxis attractants. A number of plant-derived compounds and corresponding chemotaxis sensors have been documented, however, the sensors for methanol, one of the major volatile compounds released by plants, have not been identified. Methylobacterium species are ubiquitous plant surface-symbiotic, methylotrophic bacteria. A plant-growth promoting bacterium, M. aquaticum strain 22A exhibits chemotaxis toward methanol (methylotaxis). Its genome encodes 52 methyl-accepting chemotaxis proteins (MCPs), among which we identified three MCPs (methylotaxis proteins, MtpA, MtpB, and MtpC) responsible for methylotaxis. The triple gene mutant of the MCPs exhibited no methylotaxis, slower gathering to plant tissues, and less efficient colonization on plants than the wild type, suggesting that the methylotaxis mediates initiation of plant-Methylobacterium symbiosis and engages in proliferation on plants. To examine how these MCPs are operating methylotaxis, we generated multiple gene knockouts of the MCPs, and Ca2+-dependent MxaFI and lanthanide (Ln3+)-dependent XoxF methanol dehydrogenases (MDHs), whose expression is regulated by the presence of Ln3+. MtpA was found to be a cytosolic sensor that conducts formaldehyde taxis (formtaxis), as well as methylotaxis when MDHs generate formaldehyde. MtpB contained a dCache domain and exhibited differential cellular localization in response to La3+. MtpB expression was induced by La3+, and its activity required XoxF1. MtpC exhibited typical cell pole localization, required MxaFI activity, and was regulated under MxbDM that is also required for MxaF expression. Strain 22A methylotaxis is realized by three independent MCPs, two of which monitor methanol oxidation by Ln3+-regulated MDHs, and one of which monitors the common methanol oxidation product, formaldehyde. We propose that methanol metabolism-linked chemotaxis is the key factor for the efficient colonization of Methylobacterium on plants.

Interaction between C1-microorganisms and plants: contribution to the global carbon cycle and microbial survival strategies in the phyllosphere.

C1-microorganisms that can utilize C1-compounds, such as methane and methanol, are ubiquitous in nature, and contribute to drive the global carbon cycle between two major greenhouse gases, CO2 and methane. Plants emit C1-compounds from their leaves and provide habitats for C1-microorganisms. Among C1-microorganisms, Methylobacterium spp., representative of methanol-utilizing methylotrophic bacteria, predominantly colonize the phyllosphere and are known to promote plant growth. This review summarizes the interactions between C1-mircroorganisms and plants that affect not only the fixation of C1-compounds produced by plants but also CO2 fixation by plants. We also describe our recent understanding of the survival strategy of C1-microorganisms in the phyllosphere and the application of Methylobacterium spp. to improve rice crop yield.

Phosphoregulation of the transcription factor Mxr1 plays a crucial role in the concentration-regulated methanol induction in Komagataella phaffii.

Methylotrophic yeasts can utilize methanol as the sole carbon and energy source, and the expression of their methanol-induced genes is regulated based on the environmental methanol concentration. Our understanding of the function of transcription factors and Wsc family of proteins in methanol-induced gene expression and methanol sensing is expanding, but the methanol signal transduction mechanism remains undetermined. Our study has revealed that the transcription factor KpMxr1 is involved in the concentration-regulated methanol induction (CRMI) in Komagataella phaffii (Pichia pastoris) and that the phosphorylation state of KpMxr1 changes based on methanol concentration. We identified the functional regions of KpMxr1 and determined its multiple phosphorylation sites. Non-phosphorylatable substitution mutations of these newly identified phosphorylated threonine and serine residues resulted in significant defects in CRMI. We revealed that KpMxr1 receives the methanol signal from Wsc family proteins via KpPkc1 independent of the mitogen-activated protein kinase (MAPK) cascade and speculate that the activity of KpPkc1 influences KpMxr1 phosphorylation state. We propose that the CRMI pathway from Wsc to KpMxr1 diverges from KpPkc1 and that phosphoregulation of KpMxr1 plays a crucial role in CRMI.

Siderophore for Lanthanide and Iron Uptake for Methylotrophy and Plant Growth Promotion in Methylobacterium aquaticum Strain 22A.

Methylobacterium and Methylorubrum species are facultative methylotrophic bacteria that are abundant in the plant phyllosphere. They have two methanol dehydrogenases, MxaF and XoxF, which are dependent on either calcium or lanthanides (Lns), respectively. Lns exist as insoluble minerals in nature, and their solubilization and uptake require a siderophore-like substance (lanthanophore). Methylobacterium species have also been identified as plant growth-promoting bacteria although the actual mechanism has not been well-investigated. This study aimed to reveal the roles of siderophore in Methylobacterium aquaticum strain 22A in Ln uptake, bacterial physiology, and plant growth promotion. The strain 22A genome contains an eight-gene cluster encoding the staphyloferrin B-like (sbn) siderophore. We demonstrate that the sbn siderophore gene cluster is necessary for growth under low iron conditions and was complemented by supplementation with citrate or spent medium of the wild type or other strains of the genera. The siderophore exhibited adaptive features, including tolerance to oxidative and nitrosative stress, biofilm formation, and heavy metal sequestration. The contribution of the siderophore to plant growth was shown by the repressive growth of duckweed treated with siderophore mutant under iron-limited conditions; however, the siderophore was dispensable for strain 22A to colonize the phyllosphere. Importantly, the siderophore mutant could not grow on methanol, but the siderophore could solubilize insoluble Ln oxide, suggesting its critical role in methylotrophy. We also identified TonB-dependent receptors (TBDRs) for the siderophore-iron complex, iron citrate, and Ln, among 12 TBDRs in strain 22A. Analysis of the siderophore synthesis gene clusters and TBDR genes in Methylobacterium genomes revealed the existence of diverse types of siderophores and TBDRs. Methylorubrum species have an exclusive TBDR for Ln uptake that has been identified as LutH. Collectively, the results of this study provide insight into the importance of the sbn siderophore in Ln chelation, bacterial physiology, and the diversity of siderophore and TBDRs in Methylobacterium species.

Regulation of Peroxisome Homeostasis by Post-Translational Modification in the Methylotrophic Yeast Komagataella phaffii.

The methylotrophic yeast Komagataella phaffii (synoym Pichia pastoris) can grow on methanol with an associated proliferation of peroxisomes, which are subsequently degraded by pexophagy upon depletion of methanol. Two cell wall integrity and stress response component (WSC) family proteins (Wsc1 and Wsc3) sense the extracellular methanol concentration and transmit the methanol signal to Rom2. This stimulates the activation of transcription factors (Mxr1, Trm1, and Mit1 etc.), leading to the induction of methanol-metabolizing enzymes (methanol-induced gene expression) and synthesis of huge peroxisomes. Methanol-induced gene expression is repressed by the addition of ethanol (ethanol repression). This repression is not conducted directly by ethanol but rather by acetyl-CoA synthesized from ethanol by sequential reactions, including alcohol and aldehyde dehydrogenases, and acetyl-CoA synthetase. During ethanol repression, Mxr1 is inactivated by phosphorylation. Peroxisomes are degraded by pexophagy on depletion of methanol and this event is triggered by phosphorylation of Atg30 located at the peroxisome membrane. In the presence of methanol, Wsc1 and Wsc3 repress pexophagy by transmitting the methanol signal via the MAPK cascade to the transcription factor Rlm1, which induces phosphatases involved in dephosphorylation of Atg30. Upon methanol consumption, repression of Atg30 phosphorylation is released, resulting in initiation of pexophagy. Physiological significance of these machineries involved in peroxisome homeostasis and their post-translational modification is also discussed in association with the lifestyle of methylotrophic yeast in the phyllosphere.

Autophagy as a Survival Strategy for Eukaryotic Microbes Living in the Phyllosphere.

Autophagy is an intracellular degradation process that is highly conserved among eukaryotes at the molecular level. The process was originally revealed in the budding yeast, but the physiological role of autophagy in yeast cells had remained unknown as autophagy-deficient yeast mutants did now show a clear growth phenotype in laboratory conditions. In this review, we introduce the role of autophagy in the methylotrophic yeast Candida boidinii grown on the leaf surface of Arabidopsis thaliana. Autophagy is shown to be required for proliferation in the phyllosphere, and selective autophagic pathways such as pexophagy and cytoplasm-to-vacuole targeting (Cvt) pathway are strictly regulated during both the daily cycle and the host plant life cycle. This review describes our current understanding of the role of autophagy as a survival strategy for phyllosphere fungi. Critical functions of autophagy for pathogen invasions are also discussed.

Methylotrophic bacterium-based molecular sensor for the detection of low concentrations of methanol.

Methylotrophic bacterium Methylorubrum extorquens is a promising microorganism for the production of value-added compounds from methanol. This study focused on the development of a single-cell level biosensor system that detects methanol by using the intrinsic regulatory machinery which responds to the presence of methanol in this bacterium. A green fluorescent protein (GFP) gene located downstream of the promoter region of the serine glyoxylate aminotransferase gene (Psga) or the methanol dehydrogenase subunit 1 precursor gene (PmxaF) was inserted into the chromosome of M. extorquens wild-type strain AM1. The expression of GFP upon methanol exposure was measured by spectrofluorometer and fluorescence-activated cell sorting (FACS). The strain harboring Psga-gfp emitted fluorescence only when methanol was supplied to the culture medium, while the other strain harboring PmxaF-gfp showed high basal fluorescence even in the absence of methanol. The fluorescence intensity of the Psga-gfp strain depended on a methanol concentration higher than 25 µM, and the sensitivity and dose-dependency of this strain were much higher than previous systems using Escherichia coli. The methanol-sensing properties of the engineered M. extorquens strain were comparable to those of a methylotrophic yeast-based biosensor, suggesting the usefulness of methylotrophic microorganisms as platforms for single-cell sensing of C1 compounds. The constructed methanol sensor strain, coupled with flow cytometry techniques, provides a high-throughput and highly sensitive screening method for the selection of functional methanol-producing enzymes.

Fatty acid composition of the methylotrophic yeast Komagataella phaffii grown under low- and high-methanol conditions.

In this study, we analysed the intracellular fatty acid profiles of Komagataella phaffii during methylotrophic growth. K. phaffii grown on methanol had significantly lower total fatty acid contents in the cells compared with glucose-grown cells. C18 and C16 fatty acids were the predominant fatty acids in K. phaffii, although the contents of odd-chain fatty acids such as C17 fatty acids were also relatively high. Moreover, the intracellular fatty acid composition of K. phaffii changed in response to not only carbon sources but also methanol concentrations: C17 fatty acids and C18:2 content increased significantly as methanol concentration increased, whereas C18:1 and C18:3 contents were significantly lower in methanol-grown cells. The intracellular content of unidentified compounds (Cn H2n O4 ), on the other hand, was significantly greater in cells grown on methanol. As the intracellular contents of these Cn H2n O4 compounds were significantly higher in a gene-disrupted strain for glutathione peroxidase (gpx1Δ) than in the wild-type strain, we presume that the Cn H2n O4 compounds are fatty acid peroxides. These results indicate that K. phaffii can coordinate intracellular fatty acid composition during methylotrophic growth in order to adapt to high-methanol conditions and that certain fatty acid species such as C17:0, C17:1, C17:2 and C18:2 may be related to the physiological functions by which K. phaffii adapts to high-methanol conditions.

Homeostasis of the ER redox state subsequent to proteasome inhibition.

Endoplasmic reticulum (ER) maintains within, an oxidative redox state suitable for disulfide bond formation. We monitored the ER redox dynamics subsequent to proteasome inhibition using an ER redox probe ERroGFP S4. Proteasomal inhibition initially led to oxidation of the ER, but gradually the normal redox state was recovered that further led to a reductive state. These events were found to be concomitant with the increase in the both oxidized and reduced glutathione in the microsomal fraction, with a decrease of total intracellular glutathione. The ER reduction was suppressed by pretreatment of a glutathione synthesis inhibitor or by knockdown of ATF4, which induces glutathione-related genes. These results suggested cellular adaptation of ER redox homeostasis: (1) inhibition of proteasome led to accumulation of misfolded proteins and oxidative state in the ER, and (2) the oxidative ER was then reduced by ATF4 activation, followed by influx of glutathione into the ER.

Physiology of Methylotrophs Living in the Phyllosphere.

Methanol is abundant in the phyllosphere, the surface of the above-ground parts of plants, and its concentration oscillates diurnally. The phyllosphere is one of the major habitats for a group of microorganisms, the so-called methylotrophs, that utilize one-carbon (C1) compounds, such as methanol and methane, as their sole source of carbon and energy. Among phyllospheric microorganisms, methanol-utilizing methylotrophic bacteria, known as pink-pigmented facultative methylotrophs (PPFMs), are the dominant colonizers of the phyllosphere, and some of them have recently been shown to have the ability to promote plant growth and increase crop yield. In addition to PPFMs, methanol-utilizing yeasts can proliferate and survive in the phyllosphere by using unique molecular and cellular mechanisms to adapt to the stressful phyllosphere environment. This review describes our current understanding of the physiology of methylotrophic bacteria and yeasts living in the phyllosphere where they are exposed to diurnal cycles of environmental conditions.

The methanol sensor Wsc1 and MAPK Mpk1 suppress degradation of methanol-induced peroxisomes in methylotrophic yeast.

In nature, methanol is produced during the hydrolysis of pectin in plant cell walls. Methanol on plant leaves shows circadian dynamics, to which methanol-utilizing phyllosphere microorganisms adapt. In the methylotrophic yeast Komagataella phaffii (Kp; also known as Pichia pastoris), the plasma membrane protein KpWsc1 senses environmental methanol concentrations and transmits this information to induce the expression of genes for methanol metabolism and the formation of huge peroxisomes. In this study, we show that KpWsc1 and its downstream MAPK, KpMpk1, negatively regulate pexophagy in the presence of methanol concentrations greater than 0.15%. Although KpMpk1 was not necessary for expression of methanol-inducible genes and peroxisome biogenesis, KpMpk1, the transcription factor KpRlm1 and phosphatases were found to suppress pexophagy by controlling phosphorylation of KpAtg30, the key factor in regulation of pexophagy. We reveal at the molecular level how the single methanol sensor KpWsc1 commits the cell to peroxisome synthesis and degradation according to the methanol concentration, and we discuss the physiological significance of regulating pexophagy for survival in the phyllosphere. This article has an associated First Person interview with Shin Ohsawa, joint first author of the paper.

Methanol bioeconomy: promotion of rice crop yield in paddy fields with microbial cells prepared from natural gas-derived C1 compound.

Methylotrophs, which can utilize methanol as a sole carbon source, are promising microorganisms to be exploited in a methanol-based bioeconomy, in which a variety of useful compounds are biotechnologically produced from natural gas-derived methanol. Pink-pigmented facultative methylotrophs (PPFMs) are common plant phyllospheric bacteria and are known to enhance seedling growth and total biomass of various plants. However, improvement of crop yield by inoculation of PPFMs at the field level has not been well investigated. We herein describe improvement of crop yield of several rice cultivars by foliar spraying of PPFMs. After selection of PPFM strains and rice cultivars by the in vitro seedling growth test, we further conducted paddy field experiments. The crop yield of the sake-brewing rice Oryza sativa cultivar Hakutsurunishiki was reproducibly improved in a commercial paddy field for over a 5-year period. A one-time foliar spray of PPFM cells (living or killed) or a cell wall polysaccharide fraction, after the heading date, acted in the phyllosphere and effectively improved crop yield. Our results show that the established process with PPFMs is feasible for improvement of food production in the methanol bioeconomy.

A peroxisome deficiency-induced reductive cytosol state up-regulates the brain-derived neurotrophic factor pathway.

The peroxisome is a subcellular organelle that functions in essential metabolic pathways, including biosynthesis of plasmalogens, fatty acid ß-oxidation of very-long-chain fatty acids, and degradation of hydrogen peroxide. Peroxisome biogenesis disorders (PBDs) manifest as severe dysfunction in multiple organs, including the central nervous system (CNS), but the pathogenic mechanisms in PBDs are largely unknown. Because CNS integrity is coordinately established and maintained by neural cell interactions, we here investigated whether cell-cell communication is impaired and responsible for the neurological defects associated with PBDs. Results from a noncontact co-culture system consisting of primary hippocampal neurons with glial cells revealed that a peroxisome-deficient astrocytic cell line secretes increased levels of brain-derived neurotrophic factor (BDNF), resulting in axonal branching of the neurons. Of note, the BDNF expression in astrocytes was not affected by defects in plasmalogen biosynthesis and peroxisomal fatty acid ß-oxidation in the astrocytes. Instead, we found that cytosolic reductive states caused by a mislocalized catalase in the peroxisome-deficient cells induce the elevation in BDNF secretion. Our results suggest that peroxisome deficiency dysregulates neuronal axogenesis by causing a cytosolic reductive state in astrocytes. We conclude that astrocytic peroxisomes regulate BDNF expression and thereby support neuronal integrity and function.

Methanol production by reversed methylotrophy constructed in Escherichia coli.

We constructed a reversed methylotrophic pathway that produces methanol, a promising feedstock for production of useful compounds, from fructose 6-phosphate (F6P), which can be supplied by catabolism of biomass-derived sugars including glucose, by a synthetic biology approach. Using Escherichia coli as an expression host, we heterologously expressed genes encoding methanol utilization enzymes from methylotrophic bacteria, i.e. the NAD+-dependent methanol dehydrogenase (MDH) from Bacillus methanolicus S1 and an artificial fusion enzyme of 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase from Mycobacterium gastri MB19 (HPS-PHI). We confirmed that these enzymes can catalyze reverse reactions of methanol oxidation and formaldehyde fixation. The engineered E. coli strain co-expressing MDH and HPS-PHI genes produced methanol in resting cell reactions not only from F6P but also from glucose. We successfully conferred reversed methylotrophy to E. coli and our results provide a proof-of-concept for biological methanol production from biomass-derived sugar compounds.

Engineering the expression system for Komagataella phaffii (Pichia pastoris): an attempt to develop a methanol-free expression system.

The construction of a methanol-free expression system of Komagataella phaffii (Pichia pastoris) was attempted by engineering a strong methanol-inducible DAS1 promoter using Citrobacter braakii phytase production as a model case. Constitutive expression of KpTRM1, formerly PRM1-a positive transcription regulator for methanol-utilization (MUT) genes of K. phaffii,was demonstrated to produce phytase without addition of methanol, especially when a DAS1 promoter was used but not an AOX1 promoter. Another positive regulator, Mxr1p, did not have the same effect on the DAS1 promoter, while it was more effective than KpTrmp1 on the AOX1 promoter. Removing a potential upstream repression sequence (URS) and multiplying UAS1DAS1 in the DAS1 promoter significantly enhanced the yield of C. braakii phytase with methanol-feeding, which surpassed the native AOX1 promoter by 80%. However, multiplying UAS1DAS1 did not affect the yield of methanol-free expression by constitutive KpTrm1p. Another important region to enhance the effect of KpTrm1p under a methanol-free condition was identified in the DAS1 promoter, and was termed ESPDAS1. Nevertheless, methanol-free phytase production using an engineered DAS1 promoter outperformed phytase production with the GAP promoter by 25%. Difference in regulation by known transcription factors on the AOX1 promoter and the DAS1 promoter was also illustrated.

Methylotrophic Yeasts: Current Understanding of Their C1-Metabolism and its Regulation by Sensing Methanol for Survival on Plant Leaves.

Methylotrophic yeasts, which are able to utilize methanol as the sole carbon and energy source, have been intensively studied in terms of physiological function and practical applications. When these yeasts grow on methanol, the genes encoding enzymes and proteins involved in methanol metabolism are strongly induced. Simultaneously, peroxisomes, organelles that contain the key enzymes for methanol metabolism, massively proliferate. These characteristics have made methylotrophic yeasts efficient hosts for heterologous protein production using strong and methanol-inducible gene promoters and also model organisms for the study of peroxisome dynamics. Much attention has been paid to the interaction between methylotrophic microorganisms and plants. In this chapter, we describe how methylotrophic yeasts proliferate and survive on plant leaves, focusing on their physiological functions and lifestyle in the phyllosphere. Our current understanding of the molecular basis of methanol-inducible gene expression, including methanol-sensing and its applications, is also summarized.

Community composition and methane oxidation activity of methanotrophs associated with duckweeds in a fresh water lake.

Methanotrophs are the only biological sink of the greenhouse gas methane. To understand the ecological features of methanotrophs in association with plants in the methane emitting environments, we investigated the community composition and methane oxidation of methanotrophs associated with duckweeds in a fresh water lake. Duckweeds collected from Lake Biwa, Japan over three summers showed methane consumption activity between 0.0067 and 0.89 µmol h-1 g-1 (wet weight), with the highest values occurring from the end of July to August. The methanotrophic community on duckweeds consisted primarily of γ-proteobacterial groups including the genera Methylomonas and Methylocaldum. Further analysis of co-cultures of a methanotroph isolate with sterilized duckweed revealed that the duckweed plant as well as the duckweed spent culture supernatant exerted an enhancing effect on methane oxidation. These results indicate that duckweeds not only provide a habitat for methanotrophs but also stimulate methanotrophic growth.

Evolution from covalent conjugation to non-covalent interaction in the ubiquitin-like ATG12 system.

Ubiquitin or ubiquitin-like proteins can be covalently conjugated to multiple proteins that do not necessarily have binding interfaces. Here, we show that an evolutionary transition from covalent conjugation to non-covalent interaction has occurred in the ubiquitin-like autophagy-related 12 (ATG12) conjugation system. ATG12 is covalently conjugated to its sole substrate, ATG5, by a ubiquitylation-like mechanism. However, the apicomplexan parasites Plasmodium and Toxoplasma and some yeast species such as Komagataella phaffii (previously Pichia pastoris) lack the E2-like enzyme ATG10 and the most carboxy (C)-terminal glycine of ATG12, both of which are required for covalent linkage. Instead, ATG12 in these organisms forms a non-covalent complex with ATG5. This non-covalent ATG12-ATG5 complex retains the ability to facilitate ATG8-phosphatidylethanolamine conjugation. These results suggest that ubiquitin-like covalent conjugation can evolve to a simpler non-covalent interaction, most probably when the system has a limited number of targets.

Peroxisomal Fba2p and Tal2p complementally function in the rearrangement pathway for xylulose 5-phosphate in the methylotrophic yeast Pichia pastoris.

In this work, we analyzed several genes participating in the rearrangement pathway for xylulose 5-phosphate (Xu5P) in the methylotrophic yeast Pichia pastoris (syn. Komagataella phaffii). P. pastoris has two set of genes for fructose-1,6-bisphosphate aldolase (FBA1 and FBA2) and transaldolase (TAL1 and TAL2), although there are single-copy genes for fructose-1,6-bisphosphatase (FBP1) and transketolase (TKL1), respectively. Expressions of FBP1 and TAL2 were upregulated by non-fermentative carbon sources, especially methanol was the best inducer for them, and FBA2 was induced only by methanol. On the other hand, FBA1, TAL1 and TKL1 showed constitutive expression. Strain fbp1Δ showed severe growth defect on methanol and non-fermentable carbon sources, and growth rate of strain fba2Δ in methanol medium was slightly decreased. Moreover, Fba2p and Tal2p possessed peroxisome targeting signal type 1 (PTS1), and EGFP-Fba2p and EGFP-Tal2p were found to be localized in peroxisomes. From these findings, it was suggested that Fba2p, Fbp1p and Tal2p participate in the rearrangement pathway for Xu5P in peroxisomes, and that the altered Calvin cycle and non-oxidative pentose phosphate pathway involving Tal2p function in a complementary manner.