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 a serine, reduces taxis to ethanol. Molecular dynamics simulations suggest ethanol binds McpB near residue Ala431 and mutation of this residue 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

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

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 γ peptides, which remain associated with the particle. In vitro studies previously demonstrated that the amphipathic, helical portion (amino acids 364 to 385) of γ is membrane active, suggesting a role for γ 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 γ

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

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 γ 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 γ 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 γ peptides from the virus particle, and (iii) the exposed/released γ peptides disrupt the endosomal membrane, facilitating translocation of viral RNA into the cytoplasm

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

GST pulldowns with CheD’s two binding partners (CheC and McpAc) show reduced binding by GST-CheD-F102E.

<p>Lanes 1 (GST-CheD only control), 2 (+CheC), 3 (+McpAc) and 4 (+McpCc) contain GST-CheD bound to glutathione beads. Lanes 5 (GST-CheD-F102E only control), 6 (+CheC), 7 (+McpAc) and 8 (+McpCc) contains GST-CheD-F102E bound to glutathione beads. Multiple bands of the Mcps correspond to receptor deamidation.</p

Sequence alignment of various CheDs shows that residues M101 and F102 (boxed) are conserved in species that also contain CheC.

<p>Abbreviations for species that contain CheC: Bsub, <i>Bacillus subtilis</i>; Bhal, <i>Bacillus halodurans</i>; Cace, <i>Clostridium acetobutylicum</i>; Cthe, <i>Clostridium thermocellum</i>; Dhaf, Desulfitobacterium hafniense; Oihe, Oceanobacillus iheyensis; Phor, <i>Pyrococcus horikoshii</i>; Tmar, <i>Thermotoga maritima</i>; Tten, <i>Thermoanaerobacter tengcongensis</i>. Abbreviations for species that do not contain CheC: Atum, <i>Agrobacterium tumefaciens</i>; Bbur, Borrelia burgdorferi; Ccre, Caulobacter crescentus; Neur, <i>Nitrosomonas europaea</i>; Paer, <i>Pseudomonas aeruginosa</i>; Rsph, <i>Rhodobacter sphaeroides</i>; Smel, <i>Sinorhizobium meliloti</i>; Xcam, <i>Xanthomonas campestris</i>.</p

A CheC/CheD structural model based on the <i>T. maritima</i> crystal structure (PDB ID 2F9Z) shows residues at the binding interface [<b>21</b>].

<p>Residues M101 and F102 on CheD are highlighted in cyan, and residue D149 on CheC is highlighted in yellow.</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p