Membrane contact sites regulate vacuolar fission via sphingolipid metabolism.

Membrane contact sites (MCSs) are junctures that perform important roles including coordinating lipid metabolism. Previous studies have indicated that vacuolar fission/fusion processes are coupled with modifications in the membrane lipid composition. However, it has been still unclear whether MCS-mediated lipid metabolism controls the vacuolar morphology. Here, we report that deletion of tricalbins (Tcb1, Tcb2, and Tcb3), tethering proteins at endoplasmic reticulum (ER)-plasma membrane (PM) and ER-Golgi contact sites, alters fusion/fission dynamics and causes vacuolar fragmentation in the yeast Saccharomyces cerevisiae. In addition, we show that the sphingolipid precursor phytosphingosine (PHS) accumulates in tricalbin-deleted cells, triggering the vacuolar division. Detachment of the nucleus-vacuole junction (NVJ), an important contact site between the vacuole and the perinuclear ER, restored vacuolar morphology in both cells subjected to high exogenous PHS and Tcb3-deleted cells, supporting that PHS transport across the NVJ induces vacuole division. Thus, our results suggest that vacuolar morphology is maintained by MCSs through the metabolism of sphingolipids.

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.

Contribution of a surface salt bridge to the protein stability of deep-sea Shewanella benthica cytochrome c'.

Two homologous cytochromes c', SBCP and SVCP, from deep-sea Shewanella benthica and Shewanella violacea respectively exhibit only nine surface amino acid substitutions, along with one at the N-terminus. Despite the small sequence difference, SBCP is thermally more stable than SVCP. Here, we examined the thermal stability of SBCP variants, each containing one of the nine substituted residues in SVCP, and found that the SBCP K87V variant was the most destabilized. We then determined the X-ray crystal structure of the SBCP K87V variant at a resolution of 2.1 Å. The variant retains a four-helix bundle structure similar to the wild-type, but notable differences are observed in the hydration structure around the mutation site. Instead of forming of the intrahelical salt bridge between Lys-87 and Asp-91 in the wild-type, a clathrate-like hydration around Val-87 through a hydrogen bond network with the nearby amino acid residues is observed. This network potentially enhances the ordering of surrounding water molecules, leading to an entropic destabilization of the protein. These results suggest that the unfavorable hydrophobic hydration environment around Val-87 and the inability to form the Asp-91-mediated salt bridge contribute to the observed difference in stability between SBCP and SVCP. These findings will be useful in future protein engineering for controlling protein stability through the manipulation of surface intrahelical salt bridges.

Protein-to-structure pipeline for ambient-temperature in situ crystallography at VMXi.

The utility of X-ray crystal structures determined under ambient-temperature conditions is becoming increasingly recognized. Such experiments can allow protein dynamics to be characterized and are particularly well suited to challenging protein targets that may form fragile crystals that are difficult to cryo-cool. Room-temperature data collection also enables time-resolved experiments. In contrast to the high-throughput highly automated pipelines for determination of structures at cryogenic temperatures widely available at synchrotron beamlines, room-temperature methodology is less mature. Here, the current status of the fully automated ambient-temperature beamline VMXi at Diamond Light Source is described, and a highly efficient pipeline from protein sample to final multi-crystal data analysis and structure determination is shown. The capability of the pipeline is illustrated using a range of user case studies representing different challenges, and from high and lower symmetry space groups and varied crystal sizes. It is also demonstrated that very rapid structure determination from crystals in situ within crystallization plates is now routine with minimal user intervention.

A heme pocket aromatic quadrupole modulates gas binding to cytochrome c'-ß: Implications for NO sensors.

The structural basis by which gas-binding heme proteins control their interactions with NO, CO, and O2 is fundamental to enzymology, biotechnology, and human health. Cytochromes c' (cyts c') are a group of putative NO-binding heme proteins that fall into two families: the well-characterized four alpha helix bundle fold (cyts c'-α) and an unrelated family with a large beta-sheet fold (cyts c'-ß) resembling that of cytochromes P460. A recent structure of cyt c'-ß from Methylococcus capsulatus Bath revealed two heme pocket phenylalanine residues (Phe 32 and Phe 61) positioned near the distal gas-binding site. This feature, dubbed the "Phe cap," is highly conserved within the sequences of other cyts c'-ß but is absent in their close homologs, the hydroxylamine-oxidizing cytochromes P460, although some do contain a single Phe residue. Here, we report an integrated structural, spectroscopic, and kinetic characterization of cyt c'-ß from Methylococcus capsulatus Bath complexes with diatomic gases, focusing on the interaction of the Phe cap with NO and CO. Significantly, crystallographic and resonance Raman data show that orientation of the electron-rich aromatic ring face of Phe 32 toward distally bound NO or CO is associated with weakened backbonding and higher off rates. Moreover, we propose that an aromatic quadrupole also contributes to the unusually weak backbonding reported for some heme-based gas sensors, including the mammalian NO sensor, soluble guanylate cyclase. Collectively, this study sheds light on the influence of highly conserved distal Phe residues on heme-gas complexes of cytochrome c'-ß, including the potential for aromatic quadrupoles to modulate NO and CO binding in other heme proteins.

Lentilactobacillus hilgardii H-50 strongly inhibits lipopolysaccharide-induced inflammatory responses in mouse splenocytes via its specific surface layer proteins.

AIMS: Certain lactic acid bacteria (LAB) are known to have anti-inflammatory effects; however, hiochi bacteria, which are taxonomically classified as LAB and known to spoil a traditional Japanese alcoholic beverage, have not been studied in the same context. The aim of this study is to investigate the anti-inflammatory effects of hiochi bacteria strains and the underlying mechanisms. METHODS AND RESULTS: We screened 45 strains of hiochi bacteria for anti-inflammatory effects and found that Lentilactobacillus hilgardii H-50 strongly inhibits lipopolysaccharide (LPS)-induced secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 in mouse splenocytes. This inhibition is attributed to its specific surface layer proteins (SLPs), which directly bind to LPS. CONCLUSIONS: The L. hilgardii H-50 strain exerts anti-inflammatory effects through its SLPs.

Heterologous expression and biochemical comparison of two homologous SoxX proteins of endosymbiotic Candidatus Vesicomyosocius okutanii and free-living Hydrogenovibrio crunogenus from deep-sea environments.

Candidatus Vesicomyosocius okutanii is a currently uncultured endosymbiotic bacterium of Phreagena okutanii, a clam that inhabits deep-sea vent environments. The genome of Ca. V. okutanii encodes a sulfur-oxidizing (Sox) enzyme complex, presumably generating biological energy for the host from inorganic sulfur compounds. Here, Ca. V. okutanii SoxX (VoSoxX), a mono-heme cytochrome c component of the Sox complex, was shown to be phylogenetically related to its homologous counterpart (HcSoxX) from a free-living deep-sea bacterium, Hydrogenovibrio crunogenus. Both proteins were heterologously expressed in Escherichia coli co-expressing cytochrome c maturation genes for comparative biochemical analysis. The VoSoxX recombinant had significantly lower thermal stability than HcSoxX, reflecting the difference in growth conditions of the source bacteria. The endosymbiont inhabits a mild intracellular environment, whereas the free-living bacterium dwells in a harsh environment. This study represents the first successful case of heterologous expression of genes from Ca. V. okutanii, allowing further biochemical studies of the molecular mechanism of sulfur oxidation in deep-sea environments.

Crystal structure of thermally stable homodimeric cytochrome c'-ß from Thermus thermophilus.

Cytochrome c'-ß is a heme protein that belongs to the cytochrome P460 family and consists of homodimeric subunits with a predominantly antiparallel ß-sheet fold. Here, the crystal structure of cytochrome c'-ß from the thermophilic Thermus thermophilus (TTCP-ß) is reported at 1.74 Šresolution. TTCP-ß has a typical antiparallel ß-sheet fold similar to that of cytochrome c'-ß from the moderately thermophilic Methylococcus capsulatus (MCCP-ß). The phenylalanine cap structure around the distal side of the heme is also similar in TTCP-ß and MCCP-ß, indicating that both proteins similarly bind nitric oxide and carbon monoxide, as observed spectroscopically. Notably, TTCP-ß exhibits a denaturation temperature of 117°C, which is higher than that of MCCP-ß. Mutational analysis reveals that the increased homodimeric interface area of TTCP-ß contributes to its high thermal stability. Furthermore, 14 proline residues, which are mostly located in the TTCP-ß loop regions, possibly contribute to the rigid loop structure compared with MCCP-ß, which has only six proline residues. These findings, together with those from phylogenetic analysis, suggest that the structures of Thermus cytochromes c'-ß, including TTCP-ß, are optimized for function under the high-temperature conditions in which the source organisms live.

Unexpectedly high thermostability of an NADP-dependent malic enzyme from a psychrophilic bacterium, Shewanella livingstonensis Ac10.

Psychrophilic enzymes are generally active at low temperatures, and their functions have attracted much interest in food processing, biochemical research, and chemical industry. However, their activities are usually lost above their growth temperature because of their flexible and unstable structure. Here, we unexpectedly found that a homodimeric NADP-dependent malic enzyme from a psychrophilic bacterium, Shewanella livingstonensis Ac10 (SL-ME) showed sufficient activity with 60°C treatment, similar to its counterpart from mesophilic Escherichia coli (MaeB). Consistently, SL-ME and MaeB irreversibly denatured at 71.9°C and 64.5°C, respectively. Therefore, SL-ME shows robust catalytic activity, which appears to be advantageous for its application in the bioconversion of NADP to NADPH, an essential ingredient for membrane phospholipid synthesis.

Transcriptome analysis of Chironomus sulfurosus larvae living in acidic environments: Insights into molecular mechanisms for acid tolerance.

Larvae of chironomid Chironomus sulfurosus mainly live in acidic rivers near hot springs, suggesting that they naturally select acidic environments as preferred habitats. Here we showed that C. sulfurosus larvae moved toward acidic areas and stayed alive on agar gels with a pH gradient of H2SO4, and the body fluid pH of the homogenized larvae was near neutral even acclimated under the acidic conditions, indicating mechanisms for acid tolerance. In order to gain insights into this mechanism at the molecular level, de novo RNA-seq analysis was performed on C. sulfurosus larvae. As a result, 1,208 genes were found to be significantly up-regulated in larvae acclimated at pH 2.0 compared to controls at pH 7.0. Among the up-regulated genes, ones encoding cuticle proteins, peritrophic matrix proteins, mucus-forming proteins, F-type ATPase subunits, glutathione S transferases, ß-1,3-D-glucan synthetase, hemoglobin, and cytochrome P450 were identified. This transcriptome analysis in conjunction with behavioral and biochemical assays expands our knowledge of gene expression in C. sulfurosus larvae living in acidic environments, which will provide a basis for further studies to elucidate the molecular mechanisms for acid tolerance employed by organisms in nature.

Identification of six CPC-like genes and their differential expression in leaves of tea plant, Camellia sinensis.

Tea is one of the most consumed beverages worldwide, and trichome formation in tea plant leaves impairs their commercial value. In Arabidopsis thaliana leaves, trichome formation is negatively regulated by the CPC family genes, which encode R3-type MYB transcription factors. Here, we identified six CPC-like genes in a tea plant (Camellia sinensis var. sinensis) for the first time. Simulated three-dimensional structure of the MYB domains of all the six CPC-like proteins exhibited negative charge on the surface, as observed on that of the Arabidopsis CPC protein that does not bind to DNA, indicating their similarity with regard to molecular interaction. We further found that the six CPC-like genes were differentially expressed in different developmental stages of tea leaves, and four out of the six genes were upregulated in the youngest 1st leaves, which formed more trichomes than other older leaves. Although it does not establish a causal link, the correlation between differential expression of CPC-like genes and variable trichome formation suggests that the R3-type MYB transcription factors are potential precipitating factors in affecting the value of tea leaf.

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.

Fermented date residue extract mix containing gamma-aminobutyric acid augments the immune function of mouse splenocytes.

An extract of date (fruit of a palm tree) residue plus food-grade glutamate, acetic acid, and yeast extract (date residue extract mix, DREM) has been successfully fermented with using Lactobacillus brevis JCM 1059T to produce gamma-aminobutyric acid (GABA). Here, mouse splenocytes were found to be viable when supplemented with DREM and fermented DREM containing GABA (fDREM). The addition of DREM and fDREM resulted in the secretion of tumor necrosis factor (TNF)-α from the splenocytes, fDREM being more effective than DREM. The TNF-α secretion with DREM was elevated by exogenous addition of GABA and that with fDREM was in part mediated via A-type GABA receptors. Contrary to general understanding of the suppressive effects of GABA on various biological functions, our findings suggest that GABA-containing fDREM arguments the immune function as a food and pharmaceutical material.

Expression of two glutamate decarboxylase genes in Lactobacillus brevis during gamma-aminobutyric acid production with date residue extract.

Gamma-aminobutyric acid (GABA) is produced by Lactobacillus brevis using date residue fermentation. In this study, the GABA production method was improved, for which L. brevis strain JCM 1059T was the most efficient among the four L. brevis strains examined. This was presumably due to a difference in the expression level of the gene encoding glutamate decarboxylase that catalyzes GABA synthesis.Abbreviation: GABA: gamma-aminobutyric acid.

Correction: One fold, two functions: cytochrome P460 and cytochrome c'-ß from the methanotroph Methylococcus capsulatus (Bath).

[This corrects the article DOI: 10.1039/C8SC05210G.].

One fold, two functions: cytochrome P460 and cytochrome c'-ß from the methanotroph Methylococcus capsulatus (Bath).

Nature is adept at utilising highly similar protein folds to carry out very different functions, yet the mechanisms by which this functional divergence occurs remain poorly characterised. In certain methanotrophic bacteria, two homologous pentacoordinate c-type heme proteins have been identified: a cytochrome P460 (cyt P460) and a cytochrome c'-ß (cyt cp-ß). Cytochromes P460 are able to convert hydroxylamine to nitrous oxide (N2O), a potent greenhouse gas. This reactivity is similar to that of hydroxylamine oxidoreductase (HAO), which is a key enzyme in nitrifying and methanotrophic bacteria. Cyt P460 and HAO both have unusual protein-heme cross-links, formed by a Tyr residue in HAO and a Lys in cyt P460. In contrast, cyts cp-ß (the only known cytochromes c' with a ß-sheet fold) lack this crosslink and appears to be optimized for binding non-polar molecules (including NO and CO) without enzymatic conversion. Our bioinformatics analysis supports the proposal that cyt cp-ß may have evolved from cyt P460 via a gene duplication event. Using high-resolution X-ray crystallography, UV-visible absorption, electron paramagnetic resonance (EPR) and resonance Raman spectroscopy, we have characterized the overall protein folding and active site structures of cyt cp-ß and cyt P460 from the obligate methanotroph, Methylococcus capsulatus (Bath). These proteins display a similar ß-sheet protein fold, together with a pattern of changes to the heme pocket regions and localised tertiary structure that have converted a hydroxylamine oxidizing enzyme into a gas-binding protein. Structural comparisons provide insights relevant to enzyme redesign for synthetic enzymology and engineering of gas sensor proteins. We also show the widespread occurrence of cyts cp-ß and characterise their phylogeny.

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.

Response of neutrophilic Shewanella violacea to acid stress: growth rate, organic acid production, and gene expression.

Neutrophilic Shewanella violacea is isolated from deep-sea sediments and its response to high pressure and high salinity has been investigated. Here, the pure effects of acidic pH on S. violacea physiology were examined, aiming at further understanding of its stress response mechanism. S. violacea could grow at initial pH of 5.0-7.0 without pH adjustment during the test at atmospheric pressure, and the lowest growth rate was obtained at pH 5.0. The pH of the same growth culture with an initial pH of 5.0 rose toward a neutral pH of ~ 7.0 at the exponential growth phase, indicating that S. violacea has a mechanism for acid neutralization. When S. violacea cells were grown at the fixed pH of 5.0, about five times higher concentrations of butyric and isovaleric acids were produced than at pH 7.0. The expression level of the genes encoding three enzymes for isovaleric acid synthesis from L-leucine was also found to be upregulated in S. violacea cells grown at the fixed pH of 5.0 compared with at pH 7.0 through RNA-seq analysis. Therefore, S. violacea at least produces isovaleric acid in its response to acid stress, which further deepens our understanding of the stress response mechanism inherent in this bacterium.

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.

Correction: One fold, two functions: cytochrome P460 and cytochrome c'-ß from the methanotroph Methylococcus capsulatus (Bath).

[This corrects the article DOI: 10.1039/C8SC05210G.].