Species-specific Microorganisms in Acid-tolerant Chironomus Larvae Reared in a Neutral pH Range under Laboratory Conditions: Single Dataset Analysis.

To obtain a more detailed understanding of organismal acid tolerance, the larval microbiomes of 11 Chironomus species collected from acidic or neutral pH areas in Japan and reared at pH 7-8 under laboratory conditions were systematically compared using an amplicon sequencing ana-lysis. Evenness values were lower for the larval microbiomes of acid-tolerant Chironomus cf. riparius, Chironomus fusciceps, and Chironomus sulfurosus than for eight acid-sensitive species based on an alpha diversity ana-lysis. The lower evenness observed suggested a biased abundance of microorganisms, which was consistent with the identification of Chironomus species-specific microorganisms (such as Agromyces mediolanus and Comamonas odontotermitis related bacteria) with high abundance in acid-tolerant larvae. The abundance of specific microorganisms was also high in the microbiome of acid-tolerant larvae of Chironomus acerbiphilus reared at pH 4, but not in that of acid-sensitive larvae. Based on a PICRUSt2 ana-lysis, genes involved in saccharide transport were less abundant in the microbiome of acid-tolerant larvae than in that of acid-sensitive larvae, indicating nutrient-poor acidic environments. Although these results were obtained from single datasets, acid-tolerant larvae appeared to establish Chironomus species-specific interactions with microorganisms independent of saccharides, in contrast to acid-sensitive larvae. The present study is the first step towards understanding organismal acid tolerance.

Electrochemical Enrichment and Isolation of Electrogenic Bacteria from 0.22 µm Filtrate.

Ultramicrobacteria (UMB) that can pass through a 0.22 µm filter are attractive because of their novelty and diversity. However, isolating UMB has been difficult because of their symbiotic or parasitic lifestyles in the environment. Some UMB have extracellular electron transfer (EET)-related genes, suggesting that these symbionts may grow on an electrode surface independently. Here, we attempted to culture from soil samples bacteria that passed through a 0.22 µm filter poised with +0.2 V vs. Ag/AgCl and isolated Cellulomonas sp. strain NTE-D12 from the electrochemical reactor. A phylogenetic analysis of the 16S rRNA showed 97.9% similarity to the closest related species, Cellulomonas algicola, indicating that the strain NTE-D12 is a novel species. Electrochemical and genomic analyses showed that the strain NTE-D12 generated the highest current density compared to that in the three related species, indicating the presence of a unique electron transfer system in the strain. Therefore, the present study provides a new isolation scheme for cultivating and isolating novel UMB potentially with a symbiotic relationship associated with interspecies electron transfer.

Microbiologically influenced corrosion of stainless steel independent of sulfate-reducing bacteria.

The presence and activities of microorganisms on metal surfaces can affect corrosion. Microbial communities after such corrosion incidents have been frequently analyzed, but little is known about the dynamics of microbial communities in biofilms on different types of stainless steels, such as austenitic, martensitic, and duplex stainless steels. Here, we conducted immersion experiments on 10 types of stainless steels in a freshwater environment, where microbiologically influenced corrosion was observed. During 22-month of immersion, severe localized corrosions were observed only on martensitic S40300 stainless steel. Microbial community analysis showed notable differences between non-corroded and corroded stainless steels. On the surfaces of non-corroded stainless steels, microbial communities were slowly altered and diversity decreased over time; in particular, relative abundance of Nitrospira sp. notably increased. Whereas microbial communities in corrosion products on corroded stainless steels showed low diversity; in particular, the family Beggiatoaceae bacteria, iron-oxidizing bacteria, and Candidatus Tenderia sp. were enriched. Furthermore, sulfur enrichment during localized corrosion was observed. Since there was no enrichment of sulfate-reducing bacteria, the sulfur enrichment may be derived from the presence of family Beggiatoaceae bacteria with intracellular sulfur inclusion. Our results demonstrated slow and drastic changes in microbial communities on the healthy and corroded metal surfaces, respectively, and microbial communities on the healthy metal surfaces were not affected by the composition of the stainless steel.

Novel Methanobacterium Strain Induces Severe Corrosion by Retrieving Electrons from Fe0 under a Freshwater Environment.

Methanogens capable of accepting electrons from Fe0 cause severe corrosion in anoxic conditions. In previous studies, all iron-corrosive methanogenic isolates were obtained from marine environments. However, the presence of methanogens with corrosion ability using Fe0 as an electron donor and their contribution to corrosion in freshwater systems is unknown. Therefore, to understand the role of methanogens in corrosion under anoxic conditions in a freshwater environment, we investigated the corrosion activities of methanogens in samples collected from groundwater and rivers. We enriched microorganisms that can grow with CO2/NaHCO3 and Fe0 as the sole carbon source and electron donor, respectively, in ground freshwater. Methanobacterium sp. TO1, which induces iron corrosion, was isolated from freshwater. Electrochemical analysis revealed that strain TO1 can uptake electrons from the cathode at lower than -0.61 V vs SHE and has a redox-active component with electrochemical potential different from those of other previously reported methanogens with extracellular electron transfer ability. This study indicated the corrosion risk by methanogens capable of taking up electrons from Fe0 in anoxic freshwater environments and the necessity of understanding the corrosion mechanism to contribute to risk diagnosis.

Thermal stability tuning without affecting gas-binding function of Thermochromatium tepidum cytochrome c'.

Hydrogenophilus thermoluteolus, Thermochromatium tepidum, and Allochromatium vinosum, which grow optimally at 52, 49, and 25 °C, respectively, have homologous cytochromes c' (PHCP, TTCP, and AVCP, respectively) exhibiting at least 50% amino acid sequence identity. Here, the thermal stability of the recombinant TTCP protein was first confirmed to be between those of PHCP and AVCP. Structure comparison of the 3 proteins and a mutagenesis study on TTCP revealed that hydrogen bonds and hydrophobic interactions between the heme and amino acid residues were responsible for their stability differences. In addition, PHCP, TTCP, and AVCP and their variants with altered stability similarly bound nitric oxide and carbon oxide, but not oxygen. Therefore, the thermal stability of TTCP together with PHCP and AVCP can be tuned through specific interactions around the heme without affecting their gas-binding function. These cytochromes c' will be useful as specific gas sensor proteins exhibiting a wide thermal stability range.

Accelerated glucose metabolism in hyphae-dispersed Aspergillus oryzae is suitable for biological production.

Recently, a hyphae-dispersed type of filamentous fungus Aspergillus oryzae was constructed via genetic engineering, and industrial applications are expected due to the ease of handling and to the level of protein production properties. In this study, we constructed cellulase-expressing strains using wild-type and hyphae-dispersed strains to investigate the correlation between protein productivity and metabolism. Compared with the original strain, the hyphae-dispersed cellulase-expressing strain showed elevated cellulase activity, rapid glucose consumption, increased mycelial dry weight, an increased expression of cellulase genes, and activated respiration activity. Comparative metabolomic analysis showed fewer metabolites in the glycolysis and TCA cycles in the dispersed strains than in the original strains. These results indicate that the flux of carbohydrate metabolism in the hyphae-dispersed strains is smoother than that in the original strains. Such efficient metabolic flux would contribute to efficient energy conversion and to sufficient energy supply to anabolisms, such as mycelial growth and protein production. Our findings suggest that the hyphae-dispersed strains could be a useful host not only for protein production but also for the biological production of various chemicals such as organic acids.

Thermal destabilization mechanism of cytochrome c' from psychrophilic Shewanella violacea.

Cytochrome c' is a nitric oxide (NO)-binding heme protein found in Gram negative bacteria. The thermal stability of psychrophilic Shewanella violacea cytochrome c' (SVCP) is lower than those of its homologues from other 2 psychrophilic Shewanella species, indicating that thermal destabilization mechanism for low-temperature adaptation accumulates in SVCP. In order to understand this mechanism at the amino acid level, here the stability and function of SVCP variants, modeled using the 2 homologues, were examined. The variants exhibited increased stability, and they bound NO similar to the wild type. The vulnerability as to the SVCP stability could be attributed to less hydrogen bond at the subunit interface, more flexible loop structure, and less salt bridge on the protein surface, which appear to be its destabilization mechanism. This study provides an example for controlling stability without spoiling function in psychrophilic proteins.

Pyruvate metabolism redirection for biological production of commodity chemicals in aerobic fungus Aspergillus oryzae.

Pyruvate is a central metabolite for the biological production of various chemicals. In eukaryotes, pyruvate produced by glycolysis is used in conversion to ethanol and lactate and in anabolic metabolism in the cytosol, or is transported into the mitochondria for use as a substrate in the tricarboxylic acid (TCA) cycle. In this study, we focused on controlling pyruvate metabolism in aerobic microorganisms for the biological production of various chemicals. We successfully improved productivity by redirecting pyruvate metabolism in the aerobic filamentous fungus Aspergillus oryzae via the deletion of two genes that encode pyruvate decarboxylase and mitochondrial pyruvate carriers. Production of ethanol as a major byproduct was completely inhibited, and the limited translocation of pyruvate into the mitochondria shifted the metabolism from respiration for energy conversion to the effective production of lactate or 2,3-butandiole, even under aerobic conditions. Metabolomic and transcriptomic analyses showed an emphasis on glycolysis and a repressed TCA cycle. Although the dry mycelial weights of the deletion mutants were reduced compared with those of wild type, the titer and yields of the target products were drastically increased. In particular, the redirection of pyruvate metabolism shifted from anabolism for biomass production to catabolism for the production of target chemicals. Conclusively, our results indicate that the redirection of pyruvate metabolism is a useful strategy in the metabolic engineering of aerobic microorganisms.

Differences in biochemical properties of two 5'-nucleotidases from deep- and shallow-sea Shewanella species under various harsh conditions.

Deep-sea Shewanella violacea 5'-nucleotidase (SVNTase) activity exhibited higher NaCl tolerance than that of a shallow-sea Shewanella amazonensis homologue (SANTase), the sequence identity between them being 70.4%. Here, SVNTase exhibited higher activity than SANTase with various inorganic salts, similar to the difference in their NaCl tolerance. In contrast, SVNTase activity decreased with various organic solvents, while SANTase activity was retained with the same concentrations of the solvents. Therefore, SVNTase is more robust than SANTase with inorganic salts, but more vulnerable with organic solvents. As to protein stability, SANTase was more stable against organic solvents and heat than SVNTase, which correlated with the differences in their enzymatic activities. We also found that SANTase retained higher activity for three weeks than SVNTase did in the presence of glycerol. These findings will facilitate further application of these enzymes as appropriate biological catalysts under various harsh conditions. Abbreviations: NTase: 5'-nucleotidase; SANTase: Shewanella amazonensis 5'-nucleotidase; SVNTase: Shewanella violacea 5'-nucleotidase; CD: circular dichroism.

Modified expression of multi-cellulases in a filamentous fungus Aspergillus oryzae.

Aspergillus oryzae, a filamentous fungus, can secrete large amounts of enzymes extracellularly. We constructed a genetically engineered A. oryzae that simultaneously produced cellobiohydrolase, endoglucanase, and ß-glucosidase by integrating multiple copies of the genes encoding these cellulases into fungal chromosomes. The resulting strain possessed 5-16 copies of each cellulase gene within the chromosome and showed approximately 10-fold higher activity versus single integration strains. Copy number polymorphisms were attributed to differences in flanking region sequence for the integrated gene fragments. Furthermore, we found that the P-sodM/T-glaB set demonstrated the strongest transcription levels per gene copy number. We therefore modified promoter/terminator set and cellulase gene combinations based on this polymorphism and transcription level data, with the resulting transformant showing 40-fold higher cellulolytic activity versus the single integration strain. This designed expression method could be useful for the overexpression of multiple enzymes and pathway flux control-mediated metabolic engineering in A. oryzae.

Stability of cytochromes c' from psychrophilic and piezophilic Shewanella species: implications for complex multiple adaptation to low temperature and high hydrostatic pressure.

The stability of dimeric cytochrome c' from a thermophile, as compared with that of a homologous mesophilic counterpart, is attributed to strengthened interactions around the heme and at the subunit-subunit interface, both of which are molecular interior regions. Here, we showed that interactions in the equivalent interior regions of homologous cytochromes c' from two psychrophiles, Shewanella benthica and Shewanella violacea (SBCP and SVCP, respectively) were similarly weakened as compared with those of the counterparts of psychrophilic Shewanella livingstonensis and mesophilic Shewanella amazonensis (SLCP and SACP, respectively), and consistently the stability of SVCP, SLCP, and SACP increased in that order. Therefore, the stability of cytochromes c' from the psychrophile, mesophile, and thermophile is systematically regulated in their molecular interior regions. Unexpectedly, however, the stability of SBCP was significantly higher than that of SVCP, and the former had additional molecular surface interactions. Collectively, SBCP had weakened interior interactions like SVCP did, but the former was stabilized at the molecular surface as compared with the latter, implying complex multiple adaptation of the proteins because the psychrophilic sources of SBCP and SVCP are also piezophilic, thriving in deep-sea extreme environments of low temperature and high hydrostatic pressure.

Biochemical and thermodynamic analyses of energy conversion in extremophiles.

A variety of extreme environments, characterized by extreme values of various physicochemical parameters (temperature, pressure, salinity, pH, and so on), are found on Earth. Organisms that favorably live in such extreme environments are called extremophiles. All living organisms, including extremophiles, must acquire energy to maintain cellular homeostasis, including extremophiles. For energy conversion in harsh environments, thermodynamically useful reactions and stable biomolecules are essential. In this review, I briefly summarize recent studies of extreme environments and extremophiles living in these environments and describe energy conversion processes in various extremophiles based on my previous research. Furthermore, I discuss the correlation between the biological system of electrotrophy, a third biological energy acquisition system, and the mechanism underlying microbiologically influenced corrosion. These insights into energy conversion in extremophiles may improve our understanding of the "limits of life". Abbreviations: PPi: pyrophosphate; PPase: pyrophosphatase; ITC: isothermal titration microcalorimetry; SVNTase: Shewanella violacea 5'-nucleotidase; SANTase: Shewanella amazonensis 5'-nucleotidase.

Automated image analysis tool for tumor volume growth rate to guide precision cancer therapy: EGFR-mutant non-small-cell lung cancer as a paradigm.

PURPOSE: To develop an automated analytic module for calculation of tumor growth rate from serial CT scans and to apply the module and evaluate reproducibility in a pilot cohort of advanced NSCLC patients with EGFR mutations treated with EGFR tyrosine kinase inhibitors. MATERIALS AND METHODS: The module utilized a commercially available image-processing workstation equipped with a validated tumor volume measurement tool. An automated analytic software module was programmed with the capability to record and display serial tumor volume changes and to calculate tumor volume growth rate over time and added to the workstation. The module was applied to evaluate the tumor growth rate in a pilot cohort of 24 EGFR-mutant patients treated with EGFR inhibitors, and reproducibility references as tested by two independent thoracic radiologists. RESULTS: The module analyzed chest CT scans from 24 patients (5 males, 19 females; median age: 61) with a median of 8 scans per patient, totaling 227 scans and provided a graphical display with an automated and instant calculation of tumor growth rate after the nadir volume for each patient. High inter and intraobserver agreements were noted for tumor growth rates, with concordance correlation coefficients of 0.9323 and 0.9668, respectively. Interpretation of slow versus fast tumor growth using previously identified threshold of ≤0.15/month had a perfect interobserver agreement (κ = 1.00), and an excellent intraobserver agreement (κ = 0.895). CONCLUSIONS: The present study describes the development of an image analytic module for assessing tumor growth rate and the data demonstrates the functionality and reproducibility of the module in a pilot cohort of EGFR-mutant NSCLC patients treated with EGFR-TKI. The image analytic module is an initial step for clinical translation of the tumor growth rate approach to guide cancer treatment in precision oncology.

An extracellular [NiFe] hydrogenase mediating iron corrosion is encoded in a genetically unstable genomic island in Methanococcus maripaludis.

Certain methanogens deteriorate steel surfaces through a process called microbiologically influenced corrosion (MIC). However, the mechanisms of MIC, whereby methanogens oxidize zerovalent iron (Fe0), are largely unknown. In this study, Fe0-corroding Methanococcus maripaludis strain OS7 and its derivative (strain OS7mut1) defective in Fe0-corroding activity were isolated. Genomic analysis of these strains demonstrated that the strain OS7mut1 contained a 12-kb chromosomal deletion. The deleted region, termed "MIC island", encoded the genes for the large and small subunits of a [NiFe] hydrogenase, the TatA/TatC genes necessary for the secretion of the [NiFe] hydrogenase, and a gene for the hydrogenase maturation protease. Thus, the [NiFe] hydrogenase may be secreted outside the cytoplasmic membrane, where the [NiFe] hydrogenase can make direct contact with Fe0, and oxidize it, generating hydrogen gas: Fe0 + 2 H+ → Fe2+ + H2. Comparative analysis of extracellular and intracellular proteomes of strain OS7 supported this hypothesis. The identification of the MIC genes enables the development of molecular tools to monitor epidemiology, and to perform surveillance and risk assessment of MIC-inducing M. maripaludis.

Direct and highly productive conversion of cyanobacteria Arthrospira platensis to ethanol with CaCl2 addition.

BACKGROUND: The cyanobacterium Arthrospira platensis shows promise as a carbohydrate feedstock for biofuel production. The glycogen accumulated in A. platensis can be extracted by lysozyme-degrading the peptidoglycan layer of the bacterial cell walls. The extracted glycogen can be converted to ethanol through hydrolysis by amylolytic enzymes and fermentation by the yeast Saccharomyces cerevisiae. Thus, in the presence of lysozyme, a recombinant yeast expressing α-amylase and glucoamylase can convert A. platensis directly to ethanol, which would simplify the procedure for ethanol production. However, the ethanol titer and productivity in this process are lower than in ethanol production from cyanobacteria and green algae in previous reports. RESULTS: To increase the ethanol titer, a high concentration of A. platensis biomass was employed as the carbon source for the ethanol production using a recombinant amylase-expressing yeast. The addition of lysozyme to the fermentation medium increased the ethanol titer, but not the ethanol productivity. The addition of CaCl2 increased both the ethanol titer and productivity by causing the delamination of polysaccharide layer on the cell surface of A. platensis. In the presence of lysozyme and CaCl2, ethanol titer, yield, and productivity improved to 48 g L-1, 93% of theoretical yield, and 1.0 g L-1 h-1 from A. platensis, corresponding to 90 g L-1 of glycogen. CONCLUSIONS: We developed an ethanol conversion process using a recombinant amylase-expressing yeast from A. platensis with a high titer, yield, and productivity by adding both lysozyme and CaCl2. The direct and highly productive conversion process from A. platensis via yeast fermentation could be applied to multiple industrial bulk chemicals.

Commonly stabilized cytochromes c from deep-sea Shewanella and Pseudomonas.

Two cytochromes c5 (SBcytc and SVcytc) have been derived from Shewanella living in the deep-sea, which is a high pressure environment, so it could be that these proteins are more stable at high pressure than at atmospheric pressure, 0.1 MPa. This study, however, revealed that SBcytc and SVcytc were more stable at 0.1 MPa than at higher pressure. In addition, at 0.1-150 MPa, the stability of SBcytc and SVcytc was higher than that of homologues from atmospheric-pressure Shewanella, which was due to hydrogen bond formation with the heme in the former two proteins. This study further revealed that cytochrome c551 (PMcytc) of deep-sea Pseudomonas was more stable than a homologue of atmospheric-pressure Pseudomonas aeruginosa, and that specific hydrogen bond formation with the heme also occurred in the former. Although SBcytc and SVcytc, and PMcytc are phylogenetically very distant, these deep-sea cytochromes c are commonly stabilized through hydrogen bond formation.

Stabilization of mesophilic Allochromatium vinosum cytochrome c' through specific mutations modeled by a thermophilic homologue.

AVCP cytochrome c' from mesophilic Allochromatium vinosum exhibits lower stability than a thermophilic counterpart, Hydrogenophilus thermoluteolus cytochrome c' (PHCP), in which the six specific amino acid residues that are not conserved in AVCP are responsible for its stability. Here we measured the stability of AVCP variants carrying these specific residues instead of the original AVCP ones. Among the six single AVCP variants, all of which formed a dimeric structure similar to that of the wild-type, three were successfully stabilized compared with the wild-type, while one showed lower stability than the wild-type. In addition, the most stabilized and destabilized AVCP variants could bind CO, similar to the wild-type. These results indicated that mesophilic AVCP could be stabilized through specific three mutations modeled by the thermophilic counterpart, PHCP, without changing the CO binding ability.

Future insights in fungal metabolic engineering.

Filamentous fungi exhibit versatile abilities, including organic acid fermentation, protein production, and secondary metabolism, amongst others, and thus have applications in the medical and food industries. Previous genomic analyses of several filamentous fungi revealed their further potential as host microorganisms for bioproduction. Recent advancements in molecular genetics, marker recycling, and genome editing could be used to alter transformation and metabolism, based on optimized design carbolated with computer science. In this review, we detail the current applications of filamentous fungi and describe modern molecular genetic tools that could be used to expand the role of these microorganisms in bioproduction. The present review shed light on the possibility of filamentous fungi as host microorganisms in the field of bioproduction in the future.

Pseudomonas aeruginosa cytochrome c551 denaturation by five systematic urea derivatives that differ in the alkyl chain length.

Reversible denaturation of Pseudomonas aeruginosa cytochrome c551 (PAc551) could be followed using five systematic urea derivatives that differ in the alkyl chain length, i.e. urea, N-methylurea (MU), N-ethylurea (EU), N-propylurea (PU), and N-butylurea (BU). The BU concentration was the lowest required for the PAc551 denaturation, those of PU, EU, MU, and urea being gradually higher. Furthermore, the accessible surface area difference upon PAc551 denaturation caused by BU was found to be the highest, those by PU, EU, MU, and urea being gradually lower. These findings indicate that urea derivatives with longer alkyl chains are stronger denaturants. In this study, as many as five systematic urea derivatives could be applied for the reversible denaturation of a single protein, PAc551, for the first time, and the effects of the alkyl chain length on protein denaturation were systematically verified by means of thermodynamic parameters.

Pyrophosphate hydrolysis in the extremely halophilic archaeon Haloarcula japonica is catalyzed by a single enzyme with a broad ionic strength range.

The soluble protein fraction of the extremely halophilic archaeon Haloarcula japonica exhibits substantial inorganic pyrophosphate (PPi) hydrolysis activity in the presence of 2-4 M NaCl (Wakai et al, J Biol Chem 288:29247-29251, 2013), which provides high ionic strength (2-4). In this study, much higher PPi hydrolysis activity was unexpectedly detected, even with 0 M NaCl in the presence of 100-200 mM MgSO4, providing a much lower ionic strength of 0.4-0.8, in the same protein fraction. Na+ and Mg2+ ions were required for activity under high and low ionic strength conditions, respectively. A recombinant H. japonica pyrophosphatase (HjPPase) exhibited PPi hydrolysis activity with the same broad ionic strength range, indicating that the activity associated with such a broad ionic strength range could be attributed to a single enzyme. Thus, we concluded that the broad ionic strength range of HjPPase may contribute to adaptation for both Na+ and Mg2+ which are abundant but variable in the unstable living environments of H. japonica.