Draft genome sequence of Yarrowia lipolytica NRRL Y-64008, an oleaginous yeast capable of growing on lignocellulosic hydrolysates.

Yarrowia lipolytica is an oleaginous yeast that produces high titers of fatty acid-derived biofuels and biochemicals. It can grow on hydrophobic carbon sources and lignocellulosic hydrolysates. The genome sequence of Y. lipolytica NRRL Y-64008 is reported to aid in its development as a biotechnological chassis for producing biofuels and bioproducts.

Near-complete genome sequence of Lipomyces tetrasporous NRRL Y-64009, an oleaginous yeast capable of growing on lignocellulosic hydrolysates.

Lipomyces tetrasporous is an oleaginous yeast that can utilize a variety of plant-based sugars. It accumulates lipids during growth on lignocellulosic biomass hydrolysates. We present the annotated genome sequence of L. tetrasporous NRRL Y-64009 to aid in its development as a platform organism for producing lipids and lipid-based bioproducts.

Regulation of Translation by Lysine Acetylation in Escherichia coli.

Nε-lysine acetylation is a common posttranslational modification observed in diverse species of bacteria. Aside from a few central metabolic enzymes and transcription factors, little is known about how this posttranslational modification regulates protein activity. In this work, we investigated how lysine acetylation affects translation in Escherichia coli. In multiple species of bacteria, ribosomal proteins are highly acetylated at conserved lysine residues, suggesting that this modification may regulate translation. In support of this hypothesis, we found that the addition of either of the acetyl donors acetyl phosphate and acetyl-coenzyme A inhibits translation but not transcription using an E. coli cell-free system. Further investigations using in vivo assays revealed that acetylation does not appear to alter the rate of translation elongation but, rather, increases the proportions of dissociated 30S and 50S ribosomes, based on polysome profiles of mutants or growth conditions known to promote lysine acetylation. Furthermore, ribosomal proteins are more acetylated in the disassociated 30S and 50S ribosomal subunits than in the fully assembled 70S complex. The effect of acetylation is also growth rate dependent, with disassociation of the subunits being most pronounced during late-exponential and early-stationary-phase growth-the same growth phase where protein acetylation is greatest. Collectively, our data demonstrate that lysine acetylation inhibits translation, most likely by interfering with subunit association. These results have also uncovered a new mechanism for coupling translation to the metabolic state of the cell. IMPORTANCE Numerous cellular processes are regulated in response to the metabolic state of the cell. One such regulatory mechanism involves lysine acetylation, a covalent modification involving the transfer of an acetyl group from central metabolite acetyl-coenzyme A or acetyl phosphate to a lysine residue in a protein. This posttranslational modification is known to regulate some central metabolic enzymes and transcription factors in bacteria, though a comprehensive understanding of its effect on cellular physiology is still lacking. In the present study, lysine acetylation was also found to inhibit translation in Escherichia coli by impeding ribosome association, most likely by disrupting salt bridges along the binding interface of the 30S and 50S ribosomal subunits. These results further our understanding of lysine acetylation by uncovering protein synthesis as a new target of regulation and aid in the design of bacteria for biotechnology applications where the growth conditions are known to promote lysine acetylation.

Near-Complete Genome Sequence of Zygosaccharomyces rouxii NRRL Y-64007, a Yeast Capable of Growing on Lignocellulosic Hydrolysates.

The halotolerant and osmotolerant yeast Zygosaccharomyces rouxii can produce multiple volatile compounds and has the ability to grow on lignocellulosic hydrolysates. We report the annotated genome sequence of Z. rouxii NRRL Y-64007 to support its development as a platform organism for biofuel and bioproduct production.

Integrating transcriptomic and metabolomic analysis of the oleaginous yeast Rhodosporidium toruloides IFO0880 during growth under different carbon sources.

Rhodosporidium toruloides is an oleaginous yeast capable of producing a variety of biofuels and bioproducts from diverse carbon sources. Despite numerous studies showing its promise as a platform microorganism, little is known about its metabolism and physiology. In this work, we investigated the central carbon metabolism in R. toruloides IFO0880 using transcriptomics and metabolomics during growth on glucose, xylose, acetate, or soybean oil. These substrates were chosen because they can be derived from plants. Significant changes in gene expression and metabolite concentrations were observed during growth on these four substrates. We mapped these changes onto the governing metabolic pathways to better understand how R. toruloides reprograms its metabolism to enable growth on these substrates. One notable finding concerns xylose metabolism, where poor expression of xylulokinase induces a bypass leading to arabitol production. Collectively, these results further our understanding of central carbon metabolism in R. toruloides during growth on different substrates. They may also help guide the metabolic engineering and development of better models of metabolism for R. toruloides.Key points• Gene expression and metabolite concentrations were significantly changed.• Reduced expression of xylulokinase induces a bypass leading to arabitol production.• R. toruloides reprograms its metabolism to allow growth on different substrates.

Investigating the role of the transcriptional regulator Ure2 on the metabolism of Saccharomyces cerevisiae: a multi-omics approach.

Ure2 regulates nitrogen catabolite repression in Saccharomyces cerevisiae. Deletion of URE2 induces a physiological state mimicking the nitrogen starvation and autophagic responses. Previous work has shown that deletion of URE2 increases the fermentation rate of some wine-producing strains of S. cerevisiae. In this work, we investigated the effect of URE2 deletion (ΔURE2) on the metabolism of S. cerevisiae. During growth on glucose, the ΔURE2 mutant grew at a 40% slower rate than the wild type; however, it produced ethanol at a 31% higher rate. To better under the behavior of this mutant, we performed transcriptomics and metabolomics. Analysis of the RNA sequencing results and metabolite levels indicates that the mutant strain exhibited characteristics of both nitrogen starvation and autophagy, including the upregulation of allantoin, urea, and amino acid uptake and utilization pathways and selective autophagic machinery. In addition, pyruvate decarboxylase and alcohol dehydrogenase isoforms were expressed at higher rates than the wild type. The mutant also accumulated less trehalose and glycogen, and produced more lipids. The induction of a nitrogen starvation-like state and increase in lipid production in nitrogen-rich conditions suggest that URE2 may be a promising target for metabolic engineering in S. cerevisiae and other yeasts for the production of lipids and lipid-derived compounds. KEY POINTS: • Deletion of URE2 increases ethanol and lipid production in Saccharomyces cerevisiae. • Deletion of URE2 reduces glycogen and trehalose production. • Metabolic changes mimic nitrogen starvation and autophagic response.

The Unconventional Cytoplasmic Sensing Mechanism for Ethanol Chemotaxis in Bacillus subtilis.

Motile bacteria sense chemical gradients using chemoreceptors, which consist of distinct sensing and signaling domains. The general model is that the sensing domain binds the chemical and the signaling domain induces the tactic response. Here, we investigated the unconventional sensing mechanism for ethanol taxis in Bacillus subtilis Ethanol and other short-chain alcohols are attractants for B. subtilis Two chemoreceptors, McpB and HemAT, sense these alcohols. In the case of McpB, the signaling domain directly binds ethanol. We were further able to identify a single amino acid residue, Ala431, on the cytoplasmic signaling domain of McpB that, when mutated to serine, reduces taxis to alcohols. Molecular dynamics simulations suggest that the conversion of Ala431 to serine increases coiled-coil packing within the signaling domain, thereby reducing the ability of ethanol to bind between the helices of the signaling domain. In the case of HemAT, the myoglobin-like sensing domain binds ethanol, likely between the helices encapsulating the heme group. Aside from being sensed by an unconventional mechanism, ethanol also differs from many other chemoattractants because it is not metabolized by B. subtilis and is toxic. We propose that B. subtilis uses ethanol and other short-chain alcohols to locate prey, namely, alcohol-producing microorganisms.IMPORTANCE Ethanol is a chemoattractant for Bacillus subtilis even though it is not metabolized and inhibits growth. B. subtilis likely uses ethanol to find ethanol-fermenting microorganisms to utilize as prey. Two chemoreceptors sense ethanol: HemAT and McpB. HemAT's myoglobin-like sensing domain directly binds ethanol, but the heme group is not involved. McpB is a transmembrane receptor consisting of an extracellular sensing domain and a cytoplasmic signaling domain. While most attractants bind the extracellular sensing domain, we found that ethanol directly binds between intermonomer helices of the cytoplasmic signaling domain of McpB, using a mechanism akin to those identified in many mammalian ethanol-binding proteins. Our results indicate that the sensory repertoire of chemoreceptors extends beyond the sensing domain and can directly involve the signaling domain.

In Vitro Assay for Measuring Receptor-Kinase Activity in the Bacillus subtilis Chemotaxis Pathway.

The sensing apparatus of the Bacillus subtilis chemotaxis pathway involves a complex consisting of chemoreceptors, the CheA histidine kinase, and the CheV and CheW adaptor proteins. Attractants and repellents alter the rate of CheA autophosphorylation, either by directly binding the receptors or by indirectly interacting with them through intermediate binding proteins. We describe an in vitro assay for measuring receptor-kinase activity in B. subtilis. This assay has been used to investigate the mechanism of signal transduction in B. subtilis chemotaxis and the disparate mechanisms employed by this bacterium for sensory adaptation and gradient sensing.

Ancient Regulatory Role of Lysine Acetylation in Central Metabolism.

Lysine acetylation is a common protein post-translational modification in bacteria and eukaryotes. Unlike phosphorylation, whose functional role in signaling has been established, it is unclear what regulatory mechanism acetylation plays and whether it is conserved across evolution. By performing a proteomic analysis of 48 phylogenetically distant bacteria, we discovered conserved acetylation sites on catalytically essential lysine residues that are invariant throughout evolution. Lysine acetylation removes the residue's charge and changes the shape of the pocket required for substrate or cofactor binding. Two-thirds of glycolytic and tricarboxylic acid (TCA) cycle enzymes are acetylated at these critical sites. Our data suggest that acetylation may play a direct role in metabolic regulation by switching off enzyme activity. We propose that protein acetylation is an ancient and widespread mechanism of protein activity regulation.IMPORTANCE Post-translational modifications can regulate the activity and localization of proteins inside the cell. Similar to phosphorylation, lysine acetylation is present in both eukaryotes and prokaryotes and modifies hundreds to thousands of proteins in cells. However, how lysine acetylation regulates protein function and whether such a mechanism is evolutionarily conserved is still poorly understood. Here, we investigated evolutionary and functional aspects of lysine acetylation by searching for acetylated lysines in a comprehensive proteomic data set from 48 phylogenetically distant bacteria. We found that lysine acetylation occurs in evolutionarily conserved lysine residues in catalytic sites of enzymes involved in central carbon metabolism. Moreover, this modification inhibits enzymatic activity. Our observations suggest that lysine acetylation is an evolutionarily conserved mechanism of controlling central metabolic activity by directly blocking enzyme active sites.

Metabolic Engineering of Probiotic Saccharomyces boulardii.

Saccharomyces boulardiiis a probiotic yeast that has been used for promoting gut health as well as preventing diarrheal diseases. This yeast not only exhibits beneficial phenotypes for gut health but also can stay longer in the gut than Saccharomyces cerevisiae Therefore, S. boulardiiis an attractive host for metabolic engineering to produce biomolecules of interest in the gut. However, the lack of auxotrophic strains with defined genetic backgrounds has hampered the use of this strain for metabolic engineering. Here, we report the development of well-defined auxotrophic mutants (leu2,ura3,his3, and trp1) through clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9-based genome editing. The resulting auxotrophic mutants can be used as a host for introducing various genetic perturbations, such as overexpression or deletion of a target gene, using existing genetic tools forS. cerevisiae We demonstrated the overexpression of a heterologous gene (lacZ), the correct localization of a target protein (red fluorescent protein) into mitochondria by using a protein localization signal, and the introduction of a heterologous metabolic pathway (xylose-assimilating pathway) in the genome ofS. boulardii We further demonstrated that human lysozyme, which is beneficial for human gut health, could be secreted by S. boulardii Our results suggest that more sophisticated genetic perturbations to improveS. boulardii can be performed without using a drug resistance marker, which is a prerequisite for in vivo applications using engineeredS. boulardii.

Interactions among the three adaptation systems of Bacillus subtilis chemotaxis as revealed by an in vitro receptor-kinase assay.

The Bacillus subtilis chemotaxis pathway employs three systems for sensory adaptation: the methylation system, the CheC/CheD/CheYp system, and the CheV system. Little is known in general about how these three adaptation systems contribute to chemotaxis in B. subtilis and whether they interact with one another. To further understand these three adaptation systems, we employed a quantitative in vitro receptor-kinase assay. Using this assay, we were able to determine how CheD and CheV affect receptor-kinase activity as a function of the receptor modification state. CheD was found to increase receptor-kinase activity, where the magnitude of the increase depends on the modification state of the receptor. The principal new findings concern CheV. Little was known about this protein before now. Our data suggest that this protein has two roles depending on the modification state of the receptor, one for sensory adaptation when the receptors are modified (methylated) and the other for signal amplification when they are unmodified (unmethylated). In addition, our data suggest that methylation of site 630 tunes the strength of the CheV adaptation system. Collectively, our results provide new insight regarding the integrated function of the three adaptation systems in B. subtilis.

The importance of the interaction of CheD with CheC and the chemoreceptors compared to its enzymatic activity during chemotaxis in Bacillus subtilis.

Bacillus subtilis use three systems for adaptation during chemotaxis. One of these systems involves two interacting proteins, CheC and CheD. CheD binds to the receptors and increases their ability to activate the CheA kinase. CheD also binds CheC, and the strength of this interaction is increased by phosphorylated CheY. CheC is believed to control the binding of CheD to the receptors in response to the levels of phosphorylated CheY. In addition to their role in adaptation, CheC and CheD also have separate enzymatic functions. CheC is a CheY phosphatase and CheD is a receptor deamidase. Previously, we demonstrated that CheC's phosphatase activity plays a minor role in chemotaxis whereas its ability to bind CheD plays a major one. In the present study, we demonstrate that CheD's deamidase activity also plays a minor role in chemotaxis whereas its ability to bind CheC plays a major one. In addition, we quantified the interaction between CheC and CheD using surface plasmon resonance. These results suggest that the most important features of CheC and CheD are not their enzymatic activities but rather their roles in adaptation.

Elucidation of the multiple roles of CheD in Bacillus subtilis chemotaxis.

Chemotaxis by Bacillus subtilis requires the CheD protein for proper function. In a cheD mutant when McpB was the sole chemoreceptor in B. subtilis, chemotaxis to asparagine was quite good. When McpC was the sole chemoreceptor in a cheD mutant, chemotaxis to proline was very poor. The reason for the difference between the chemoreceptors is because CheD deamidates Q609 in McpC and does not deamidate McpB. When mcpC-Q609E is expressed as the sole chemoreceptor in a cheD background, chemotaxis is almost fully restored. Concomitantly, in vitro McpC activates the CheA kinase poorly, whereas McpC-Q609E activates it much more. Moreover, CheD, which activates chemoreceptors, binds better to McpC-Q609E compared with unmodified McpC. Using hydroxyl radical susceptibility in the presence or absence of CheD, the most likely sites of CheD binding were the modification sites where CheD, CheB and CheR carry out their catalytic activities. Thus, CheD appears to have two separate roles in B. subtilis chemotaxis - to bind to chemoreceptors to activate them as part of the CheC/CheD/CheYp adaptation system and to deamidate selected residues to activate the chemoreceptors and enable them to mediate amino acid chemotaxis.

Attractant binding induces distinct structural changes to the polar and lateral signaling clusters in Bacillus subtilis chemotaxis.

Bacteria employ a modified two-component system for chemotaxis, where the receptors form ternary complexes with CheA histidine kinases and CheW adaptor proteins. These complexes are arranged in semi-ordered arrays clustered predominantly at the cell poles. The prevailing models assume that these arrays are static and reorganize only locally in response to attractant binding. Recent studies have shown, however, that these structures may in fact be much more fluid. We investigated the localization of the chemotaxis signaling arrays in Bacillus subtilis using immunofluorescence and live cell fluorescence microscopy. We found that the receptors were localized in clusters at the poles in most cells. However, when the cells were exposed to attractant, the number exhibiting polar clusters was reduced roughly 2-fold, whereas the number exhibiting lateral clusters distinct from the poles increased significantly. These changes in receptor clustering were reversible as polar localization was reestablished in adapted cells. We also investigated the dynamic localization of CheV, a hybrid protein consisting of an N-terminal CheW-like adaptor domain and a C-terminal response regulator domain that is known to be phosphorylated by CheA, using immunofluorescence. Interestingly, we found that CheV was localized predominantly at lateral clusters in unstimulated cells. However, upon exposure to attractant, CheV was found to be predominantly localized to the cell poles. Moreover, changes in CheV localization are phosphorylation-dependent. Collectively, these results suggest that the chemotaxis signaling arrays in B. subtilis are dynamic structures and that feedback loops involving phosphorylation may regulate the positioning of individual proteins.

Low endocytic pH and capsid protein autocleavage are critical components of Flock House virus cell entry.

The process by which nonenveloped viruses cross cell membranes during host cell entry remains poorly defined; however, common themes are emerging. Here, we use correlated in vivo and in vitro studies to understand the mechanism of Flock House virus (FHV) entry and membrane penetration. We demonstrate that low endocytic pH is required for FHV infection, that exposure to acidic pH promotes FHV-mediated disruption of model membranes (liposomes), and particles exposed to low pH in vitro exhibit increased hydrophobicity. In addition, FHV particles perturbed by heating displayed a marked increase in liposome disruption, indicating that membrane-active regions of the capsid are exposed or released under these conditions. We also provide evidence that autoproteolytic cleavage, to generate the lipophilic gamma peptide (4.4 kDa), is required for membrane penetration. Mutant, cleavage-defective particles failed to mediate liposome lysis, regardless of pH or heat treatment, suggesting that these particles are not able to expose or release the requisite membrane-active regions of the capsid, namely, the gamma peptides. Based on these results, we propose an updated model for FHV entry in which (i) the virus enters the host cell by endocytosis, (ii) low pH within the endocytic pathway triggers the irreversible exposure or release of gamma peptides from the virus particle, and (iii) the exposed/released gamma peptides disrupt the endosomal membrane, facilitating translocation of viral RNA into the cytoplasm.

Dissecting the functional domains of a nonenveloped virus membrane penetration peptide.

Recent studies have established that several nonenveloped viruses utilize virus-encoded lytic peptides for host membrane disruption. We investigated this mechanism with the "gamma" peptide of the insect virus Flock House virus (FHV). We demonstrate that the C terminus of gamma is essential for membrane disruption in vitro and the rescue of immature virus infectivity in vivo, and the amphipathic N terminus of gamma alone is not sufficient. We also show that deletion of the C-terminal domain disrupts icosahedral ordering of the amphipathic helices of gamma in the virus. Our results have broad implications for understanding membrane lysis during nonenveloped virus entry.

Rescue of maturation-defective flock house virus infectivity with noninfectious, mature, viruslike particles.

The infectivity of flock house virus (FHV) requires autocatalytic maturation cleavage of the capsid protein at residue 363, liberating the C-terminal 44-residue gamma peptides, which remain associated with the particle. In vitro studies previously demonstrated that the amphipathic, helical portion (amino acids 364 to 385) of gamma is membrane active, suggesting a role for gamma in RNA membrane translocation during infection. Here we show that the infectivity of a maturation-defective mutant of FHV can be restored by viruslike particles that lack the genome but undergo maturation cleavage. We propose that the colocalization of the two defective particle types in an entry compartment allows the restoration of infectivity by gamma.

Morphological changes in the T=3 capsid of Flock House virus during cell entry.

We report the identification and characterization of a viral intermediate formed during infection of Drosophila cells with the nodavirus Flock House virus (FHV). We observed that even at a very low multiplicity of infection, only 70% of the input virus stayed attached to or entered the cells, while the remaining 30% of the virus eluted from cells after initial binding. The eluted FHV particles did not rebind to Drosophila cells and, thus, could no longer initiate infection by the receptor-mediated entry pathway. FHV virus-like particles with the same capsid composition as native FHV but containing cellular RNA also exhibited formation of eluted particles when incubated with the cells. A maturation cleavage-defective mutant of FHV, however, did not. Compared to naïve FHV particles, i.e., particles that had never been incubated with cells, eluted particles showed an acid-sensitive phenotype and morphological alterations. Furthermore, eluted particles had lost a fraction of the internally located capsid protein gamma. Based on these results, we hypothesize that FHV eluted particles represent an infection intermediate analogous to eluted particles observed for members of the family Picornaviridae.