Assessing MAPK contributions to SKLC phosphorylation.

<p>HA immunoblots of lysates from embryos programmed to express SKLC^WT with the <i>69B-Gal4</i> driver (control), or with either loss-of-function (RNAi knockdown or dominant negative) or gain-of-function (wildtype or constitutive active) constructs for the p38 or JNK pathways. Each genotype was assayed without and with heat shock (HS) treatment to test the requirement for heat-induced phosphorylation. The intensity (grey value) profiles are shown with peaks (leftward) representing the upper and lower bands of SKLC protein.</p

Longevity of <i>slpr</i> mutant and transgenic flies.

<p>Survival of adult flies was monitored daily. Statistical analysis was performed using the log rank test. Cohort sizes and p-values were as follows. Upper graph: <i>w¹¹¹⁸</i> males (n = 194) vs. <i>slpr^BS06</i> males (n = 183) p<0.0001, <i>w¹¹¹⁸</i> females (n = 153) vs. <i>slpr^BS06</i> females (n = 115) p<0.0001, <i>w¹¹¹⁸</i> females vs. <i>w¹¹¹⁸/slpr^BS06</i> females (n = 157) p = n.s. Lower graph: Data for males and females is combined. <i>+</i> (n = 336) vs. <i>SlprWT</i> (n = 184) p<0.0001, <i>+</i> vs. <i>PXAP</i> (n = 134) or <i>SlprAAA</i> (n = 280) or <i>PXEP</i> (n = 368) were not significantly different. <i>PXAP</i> vs. <i>PXEP</i> p = 0.0013.</p

Mutant PXSP transgenes retain signaling function in development.

<p>(A) Immunofluorescence staining for Fasciclin 3 (red) in the ectoderm allowed visualization of the progress of dorsal closure in embryos expressing the indicated HA-tagged Slpr transgenes (green) with <i>pnr-Gal4</i> compared to a control <i>w¹¹¹⁸</i> (Aa) embryo. Expression of SlprAAA (Ac) causes detachment of the dorsal ectoderm anteriorly and closure defects, whereas overexpression of SlprWT, PXAP, or PXEP, promoted closure of the epidermis at the dorsal midline (Ab,d,e). Images are dorsolateral views of stage 16/17 embryos with anterior left. (B) JNK target gene expression, monitored by the <i>puc-lacZ</i> reporter, was upregulated in the <i>pnr</i> domain of the embryonic dorsal ectoderm upon expression of all of the indicated Slpr transgenes (Bb,d,e) except the dominant negative SlprAAA (Bc) compared to a control embryo without a Slpr transgene (Ba). Images are dorsolateral views of stage 14/15 embryos, with anterior left. (C) The degree of rescue of <i>slpr^BS06/Y</i> mutants to adulthood, with expression of the indicated Slpr transgenes under <i>arm-Gal4</i> control, is displayed as the percentage relative to <i>FM7/Y</i> siblings in comparison to a no transgene control. Error bars show s.d. Significant p-values are given, based on Students <i>t</i>-test. The total numbers of flies scored are shown above each bar and comprise data from at least three independent transgenic lines per construct. (D) Thorax closure was used to monitor variable levels of JNK signaling during metamorphosis. Blue dots and lines mark the positions of the posterior dorsocentral bristles that flank the dorsal midline. Yellow dots mark scutellar bristles (left of midline only) on the scutellum. SlprWT overexpression with <i>pnr-Gal4</i> upregulated JNK activity, which was evident as a narrowed scutellum and/or loss of scutellar bristles (Db) compared to control (Da). Conversely, SlprAAA overexpression impaired JNK-dependent thorax closure resulting in a cleft thorax (note blue dots are farther apart) and widened scutellum. Overexpression of both PXAP and PXEP led to reduced scutellum and loss of scutellar bristles (Dd,e); PXEP resulted in consistently more severe phenotypes. Dorsal views of adult thorax, anterior up.</p

Slpr domain architecture and putative phosphorylation at a conserved PXSP motif.

<p>(A) The wildtype Slpr protein (SlprWT) consists of an N-terminal SH3 domain, the catalytic kinase domain, a leucine zipper (LZ) domain, a CRIB domain, and a long C-terminus. Full-length wildtype, mutant, or truncated constructs are indicated by solid lines below. SKLC and SK, encode either the four main domains or the SH3 and kinase only, respectively. SKLC^mut contains a kinase mutation, D314Y, while SKLC²³¹³ is truncated immediately downstream of the CRIB domain, lacking eight amino acids spanning the PGSP site. SlprAAA contains three mutations (T287A, S291A, and T295A) in the kinase activation loop. PXAP and PXEP are full-length forms of the Slpr protein with an S512A or S512E mutation that either abolishes or mimics phosphorylation, respectively. Each construct has a C-terminal HA tag. The sizes of encoded proteins (excluding the tag) are indicated. (B) Multisequence alignment of insect, hydra, and mammalian MLK family proteins spanning the proline and basic amino acid linker region, the CRIB domain, and downstream conserved PXSP motif. The degree of sequence conservation among the orthologues is indicated by the intensity of highlighting. A phosphorylated form of the peptide in Slpr (Dme MLK) that has been recovered in proteomic studies is boxed. The serine residue targeted for phosphorylation is highlighted in red. (C–D) The different Slpr SKLC proteins and SK were translated and labeled <i>in vitro</i> with ³⁵S-methionine in a wheat germ lysate. SKLC^WT and SKLC^mut proteins migrated as doublets by SDS-PAGE. Lambda phosphatase treatment removed the upper band of the doublet of SKLC^WT and SKLC^mut indicative of a phosphorylated form (C). In contrast, the SK and SKLC²³¹³ proteins, which lack the PXSP motif, migrated as single bands (D).</p

Tales of diversity: Genomic and morphological characteristics of forty-six <i>Arthrobacter</i> phages

<div><p>The vast bacteriophage population harbors an immense reservoir of genetic information. Almost 2000 phage genomes have been sequenced from phages infecting hosts in the phylum Actinobacteria, and analysis of these genomes reveals substantial diversity, pervasive mosaicism, and novel mechanisms for phage replication and lysogeny. Here, we describe the isolation and genomic characterization of 46 phages from environmental samples at various geographic locations in the U.S. infecting a single <i>Arthrobacter</i> sp. strain. These phages include representatives of all three virion morphologies, and Jasmine is the first sequenced podovirus of an actinobacterial host. The phages also span considerable sequence diversity, and can be grouped into 10 clusters according to their nucleotide diversity, and two singletons each with no close relatives. However, the clusters/singletons appear to be genomically well separated from each other, and relatively few genes are shared between clusters. Genome size varies from among the smallest of siphoviral phages (15,319 bp) to over 70 kbp, and G+C contents range from 45–68%, compared to 63.4% for the host genome. Although temperate phages are common among other actinobacterial hosts, these <i>Arthrobacter</i> phages are primarily lytic, and only the singleton Galaxy is likely temperate.</p></div

Genome organization of <i>Arthrobacter</i> phage Jawnski, Cluster AO.

<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180517#pone.0180517.g005" target="_blank">Fig 5</a> for details.</p

Genome organization of <i>Arthrobacter</i> phage Circum, Cluster AM.

<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180517#pone.0180517.g005" target="_blank">Fig 5</a> for details.</p

Genome organization of <i>Arthrobacter</i> phage Amigo, Cluster AQ.

<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180517#pone.0180517.g005" target="_blank">Fig 5</a> for details.</p

Genome organization of <i>Arthrobacter</i> phage Maggie, Cluster AN.

<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180517#pone.0180517.g005" target="_blank">Fig 5</a> for details.</p

Genome organization of <i>Arthrobacter</i> phage Galaxy, Singleton.

<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180517#pone.0180517.g005" target="_blank">Fig 5</a> for details.</p