Identification of NpdA as the protein forming the surface layer in Paracidovorax citrulli and evidence of its occurrence as a surface layer protein in diverse genera of the Betaproteobacteria and Gammaproteobacteria.

The phytopathogen Paracidovorax citrulli possesses an ortholog of a newly identified surface layer protein (SLP) termed NpdA but has not been reported to produce a surface layer (S-layer). This study had two objectives. First, to determine if P. citrulli formed an NpdA-based S-layer and, if so, assess the effects of S-layer formation on virulence, production of nanostructures termed nanopods, and other phenotypes. Second, to establish the distribution of npdA orthologs throughout the Pseudomonadota and examine selected candidate cultures for physical evidence of S-layer formation. Formation of an NpdA-based S-layer by P. citrulli AAC00-1 was confirmed by gene deletion mutagenesis (ΔnpdA), proteomics, and cryo-electron microscopy. There were no significant differences between the wild-type and mutant in virulence assays with detached watermelon fruit. Nanopods contiguous with S-layers of multiple biofilm cells were visualized by transmission electron microscopy. Orthologs of npdA were identified in 62 Betaproteobacteria species and 49 Gammaproteobacteria species. In phylogenetic analyses, NpdA orthologs largely segregated into distinct groups. Cryo-electron microscopy imaging revealed an NpdA-like S-layer in all but one of the 16 additional cultures examined. We conclude that NpdA represents a new family of SLP, forming an S-layer in P. citrulli and other Pseudomonadota. While the S-layer did not contribute to virulence in watermelon fruit, a potential role of the P. citrulli S-layer in another dimension of pathogenesis cannot be ruled out. Lastly, formation of cell-bridging nanopods in biofilms is a new property of S-layers; it remains to be determined if nanopods can mediate intercellular movement of materials.

Early-onset sleep defects in Drosophila models of Huntington's disease reflect alterations of PKA/CREB signaling.

Huntington's disease (HD) is a progressive neurological disorder whose non-motor symptoms include sleep disturbances. Whether sleep and activity abnormalities are primary molecular disruptions of mutant Huntingtin (mutHtt) expression or result from neurodegeneration is unclear. Here, we report Drosophila models of HD exhibit sleep and activity disruptions very early in adulthood, as soon as sleep patterns have developed. Pan-neuronal expression of full-length or N-terminally truncated mutHtt recapitulates sleep phenotypes of HD patients: impaired sleep initiation, fragmented and diminished sleep, and nighttime hyperactivity. Sleep deprivation of HD model flies results in exacerbated sleep deficits, indicating that homeostatic regulation of sleep is impaired. Elevated PKA/CREB activity in healthy flies produces patterns of sleep and activity similar to those in our HD models. We were curious whether aberrations in PKA/CREB signaling were responsible for our early-onset sleep/activity phenotypes. Decreasing signaling through the cAMP/PKA pathway suppresses mutHtt-induced developmental lethality. Genetically reducing PKA abolishes sleep/activity deficits in HD model flies, restores the homeostatic response and extends median lifespan. In vivo reporters, however, show dCREB2 activity is unchanged, or decreased when sleep/activity patterns are abnormal, suggesting dissociation of PKA and dCREB2 occurs early in pathogenesis. Collectively, our data suggest that sleep defects may reflect a primary pathological process in HD, and that measurements of sleep and cAMP/PKA could be prodromal indicators of disease, and serve as therapeutic targets for intervention.

dCREB2-mediated enhancement of memory formation.

CREB-responsive transcription has an important role in adaptive responses in all cells and tissue. In the nervous system, it has an essential and well established role in long-term memory formation throughout a diverse set of organisms. Activation of this transcription factor correlates with long-term memory formation and disruption of its activity interferes with this process. Most convincingly, augmenting CREB activity in a number of different systems enhances memory formation. In Drosophila, a sequence rearrangement in the original transgene used to enhance memory formation has been a source of confusion. This rearrangement prematurely terminates translation of the full-length protein, leaving the identity of the "enhancing molecule" unclear. In this report, we show that a naturally occurring, downstream, in-frame initiation codon is used to make a dCREB2 protein off of both transgenic and chromosomal substrates. This protein is a transcriptional activator and is responsible for memory enhancement. A number of parameters can affect enhancement, including the short-lived activity of the activator protein, and the time-of-day when induction and behavioral training occur. Our results reaffirm that overexpression of a dCREB2 activator can enhance memory formation and illustrate the complexity of this behavioral enhancement.