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.

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 ␥ 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 ␥ 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 ␥ peptides from the virus particle, and (iii) the exposed/released ␥ peptides disrupt the endosomal membrane, facilitating translocation of viral RNA into the cytoplasm.Flock House virus (FHV), a nonenveloped, positive-sense RNA virus, has been employed as a model system in several important studies to address a wide range of biological questions (reviewed in reference 55). FHV has been instrumental in understanding virus structure and assembly (17,19,45), RNA replication (2,3,37), and specific packaging of the genome (33,44,53,54). Studies of FHV infection in Drosophila melanogaster flies have provided valuable information about the antiviral innate immune response in invertebrate hosts (29,57). FHV is also used in nanotechnology applications as an epitope-presenting platform to develop novel vaccines and medical therapies (31,48). In this report, we use FHV as a model system to further elucidate the means by which nonenveloped viruses enter host cells and traverse cellular membranes.

Saccharomyces boulardii is 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. boulardii is 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 for S. 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 of S. 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 improve S. boulardii can be performed without using a drug resistance marker, which is a prerequisite for in vivo applications using engineered S. boulardii.

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.The presence of membrane lytic peptides in many nonenveloped viruses is well established (3, 16), but how these peptides are deployed from the virus capsid during host cell entry and disrupt membranes remains unclear. These peptides are typically generated by a postassembly proteolytic processing event (1, 11) and are exposed from a previously buried position during conformational alterations in the capsid triggered by host cell conditions (2, 18). Flock House virus (FHV), an insect nodavirus, contains a 4-kDa peptide called "gamma" (␥), which shares many of the characteristics of other nonenveloped virus lytic peptides (3). The FHV capsid is made from 180 copies of a single-coat protein (␣) enclosing a single-stranded bipartite RNA genome (9). Gamma is generated by the autocatalytic cleavage of ␣ during virus maturation (␣ 3 ␤ ϩ ␥) (15), remains localized in the capsid interior (9) with occasional externalization or "breathing" (6), and is exposed under low-pH conditions in the endosomes during entry (Odegard et al., submitted for publication). Covalently independent gamma is necessary for virus infection, since maturation-defective FHV (D75N/N363T FHV), which does not undergo the autocleavage of ␣, is not infectious (15,17). The N-terminal ϳ21 residues of gamma (corresponding to residues 364 to 384 of ␣) constitute an amphipathic helix which can disrupt membranes in vitro when synthetically produced (4, 5) and is recognized as the host membrane-interacting region of FHV during entry. The hydrophobic, ϳ23-residue-long C terminus of gamma, especially certain phenylalanine residues (at positions 402, 405, and 407), is responsible for specifically packaging viral RNA into capsids during assembly (14).It was recently demonstrated that a supply of full-length gamma from noninfectious virus-like particles (VLPs) of FHV (13) during entry can restore infectivity to maturation-defective FHV (17), suggesting that gamma can function in trans to mediate access into host cells. This trans-complementation assay (17) was utilized to provide a quantitative readout of the effect of gamma mutations specifically on virus entry and to thus assess the region(s) of gamma required during virus entry. To determine the minimal sequence of gamma required for trans-complementation, FHV VLPs were produced that included the first 384, 390, or 395 amino acids of capsid protein ...

No abstract

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.Many motile bacteria employ for chemotaxis a modified two-component system to sense and respond to chemicals, where the receptors form ternary complexes with the CheA histidine kinase and the CheW adaptor protein (1, 2). The clustering of these ternary complexes into semi-ordered hexagonal lattices has been documented in multiple species (3) and is presumably conserved in all chemotactic bacteria where the three proteins are found. These arrays are thought to amplify the response to attractant binding (4,5). A number of models have specifically proposed that cooperative interactions between the receptors within these arrays enable bacteria to sense small differences in the number of attractant-bound receptors over a wide range of concentrations (see Ref. 6).Multiple studies have investigated the structure and molecular determinants of these clusters (e.g. Refs. 7 and 8) along with their role in signal transduction. In Escherichia coli, the receptors form mixed trimers of receptor homodimers. These trimers are believed to form the basic building blocks for the larger clusters, which range in size from tens to thousands of receptors (9). These clusters are found predominantly at the cell poles, although they are also found along the lateral length of the cell. Attractant binding, which inhibi...

Summary 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.

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.Nonenveloped viruses are stable protein complexes designed to protect and transport the viral genome from cell to cell. During assembly and disassembly, these complexes undergo transitions through meta-stable intermediates. Metastable assembly intermediates known as provirions have previously been identified and characterized for several viruses (9, 15, 19), but less is known about the intermediates formed during viral cell entry. Members of the family Picornaviridae, including poliovirus, human rhinovirus, and coxsackievirus B3, are known to form "eluted particle" intermediates during cell entry (3,8,11,14,22,(27)(28)(29). Eluted particles are virions that, after initial binding to their cognate receptor, have dissociated from the receptor in an altered form. They have lost the internal capsid protein VP4 and sediment at a decreased rate on sucrose gradients. They also display altered antigenic properties, show increased protease susceptibility, and, most importantly, are no longer able to reattach to their receptor (11,13,14,17,27).Here we report that Flock House virus (FHV), a member of the family Nodaviridae, also forms eluted particles during the initial stages of viral infection. FHV is a nonenveloped icosahedral insect virus with a bipartite positive-strand RNA genome (for a review, see references 2 and 37). Its life cycle is confined to the cytoplasm of infected cells. FHV has a wellcharacterized Tϭ3 capsid that is initially assembled from multiple subunits of the single structural precursor protein alpha (16,19). Following assembly, alpha protein undergoes a maturation cleavage, which gives rise to the major coat protein beta and a small peptide, gamma, which remains associated with mature virions and is located inside the virus particle near the packaged RNA (16,19). Previous results have shown that the C-terminal...