bPoint mutations with unclear molecular mechanisms are often associated with vancomycin resistance in Staphylococcus aureus. Here, we observed that the walK (G223D) mutation caused decreased expression of genes associated with cell wall metabolism, decreased autolytic activity, thickened cell walls, and reduced vancomycin susceptibility. A phosphorylation assay showed that WalK (G223D) exhibited reduced autophosphorylation, which led to reduced phosphorylation of WalR. An electrophoretic mobility shift assay indicated that WalK (G223D)-phosphorylated WalR had a reduced capacity to bind to the atlA promoter. (2), graRS (3, 4), and walKR (5, 6), were shown to contribute to the development of VISA (5, 7). However, the molecular mechanisms have been incompletely defined (8). WalK is a sensor kinase of the WalKR two-component regulatory system (9-11), and walK mutations across the spectrum of the domains that contribute to two-component regulatory function have been found in many clinical VISA strains isolated from various countries and laboratory-derived VISA (5, 6). Nevertheless, the same mutations are not found in both clinical and laboratoryderived VISA strains. Thus, a key question is whether the mechanism of laboratory-derived VISA strains is analogous to that of clinical VISA strains. WThe laboratory-derived VISA strain, designated SV-1, was selected by serial passage of the susceptible S. aureus strain MW2 through progressively increasing concentrations of vancomycin according to a previously described protocol (5). To identify the genetic changes that confer vancomycin resistance, whole-genome sequencing of wild-type MW2 and SV-1 was completed at the National Center for Gene Research using the paired-end sequencing of Solexa. Five mutations were identified (Table 1) and further confirmed by PCR and sequencing (see the supplemental material). Among the mutations, a single-nucleotide polymorphism within walK (conferring the G223D amino acid change) in SV-1 was also found in the clinical VISA strain JKD6008 (6), supporting the validity of our approach in identifying clinically relevant resistance mechanisms and suggesting that this point mutation probably plays an important role in reduced vancomycin susceptibilities in clinical S. aureus isolates.To determine the effect of the walK mutation, allelic replacement was performed to generate the walK mutant using the vector pBTs, which was derived from pBT2 and pKOR1 (12, 13). To construct pBTs, the segment containing antisense secY, which can inhibit colony formation on agar plates, was cloned into pBT2, and the fragment containing the walK point mutation was cloned into pBTs (see the supplemental material). Antibiotic susceptibilities of the walK mutant were evaluated by determining the MICs of vancomycin and daptomycin using

Highlights d Fbf1 tm1a/tm1a mice exhibit ''healthy obesity'' with healthy and beiging white fat tissue d FBF1 controls the beiging program via a cilia-specific, AKAP9-dependent, PKA signaling d FBF1 governs the adipogenic program via Hh signaling d Fbf1 tm1a/tm1a mice are protected from diabetes and show reduced risk of dying prematurely

bStaphylococcus aureus is an important Gram-positive pathogen responsible for numerous diseases ranging from localized skin infections to life-threatening systemic infections. The virulence of S. aureus is essentially determined by a wide spectrum of factors, including cell wall-associated proteins and secreted toxins that are precisely controlled in response to environmental changes. GGDEF domain protein from Staphylococcus (GdpS) is the only conserved staphylococcal GGDEF domain protein that is involved not in c-di-GMP synthesis but in the virulence regulation of S. aureus NCTC8325. Our previous study showed that the inactivation of gdpS generates an extensive change of virulence factors together with, in particular, a major Spa (protein A) surface protein. As reported, sarS is a direct positive regulator of spa. The decreased transcript levels of sarS in the gdpS mutant compared with the parental NCTC8325 strain suggest that gdpS affects spa through interaction with sarS. In this study, site mutation and complementary experiments showed that the translation product of gdpS was not involved in the regulation of transcript levels of sarS. We found that gdpS functioned through direct RNA-RNA base pairing with the 5= untranslated region (5=UTR) of sarS mRNA and that a putative 18-nucleotide region played a significant role in the regulatory process. Furthermore, the mRNA half-life analysis of sarS in the gdpS mutant showed that gdpS positively regulates the mRNA levels of sarS by contributing to the stabilization of sarS mRNA, suggesting that gdpS mRNA may regulate spa expression in an RNA-dependent pathway.

Mitochondrial disorders arise from defects in nuclear genes encoding enzymes of oxidative metabolism. Mutations of metabolic enzymes in somatic tissues can cause cancers due to oncometabolite accumulation. Paraganglioma and pheochromocytoma are examples, whose etiology and therapy are complicated by the absence of representative cell lines or animal models. These tumors can be driven by loss of the tricarboxylic acid cycle enzyme succinate dehydrogenase. We exploit the relationship between succinate accumulation, hypoxic signaling, egg-laying behavior, and morphology in C. elegans to create genetic and pharmacological models of succinate dehydrogenase loss disorders. With optimization, these models may enable future high-throughput screening efforts.

Mutation of ciliopathy protein HYLS1 causes the perinatal lethal hydrolethalus syndrome (HLS), yet the underlying molecular etiology and pathogenesis remain elusive. Here, we reveal unexpected mechanistic insights into the role of mammalian HYLS1 in regulating primary cilia. HYLS1 is recruited to the ciliary base via a direct interaction with the type Iγ phosphatidylinositol 4-phosphate [PI(4)P] 5-kinase (PIPKIγ). HYLS1 activates PIPKIγ by interrupting the autoinhibitory dimerization of PIPKIγ, which thereby expedites depletion of centrosomal PI(4)P to allow axoneme nucleation. HYLS1 deficiency interrupts the assembly of ciliary NPHP module and agonist-induced ciliary exit of β-arrestin, which, in turn, disturbs the removal of ciliary Gpr161 and activation of hedgehog (Hh) signaling. Consistent with this model of pathogenesis, the HLS mutant HYLS1D211G supports ciliogenesis but not activation of Hh signaling. These results implicate mammalian HYLS1 as a multitasking protein that facilitates ciliogenesis and ciliary signaling by coordinating with the ciliary lipid kinase PIPKIγ.

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